Université de Montréal 'Evo1ution ofC2H2-Zinc finger genes ...

Université de Montréal

‘Evo1ution ofC2H2-Zinc finger genes in mammalian genomes”

par

“Hamsa Dhwani Tadepally”

“Département de Biochimie”

“Faculté de Médecine”

Thèse présentée à la Faculté des études supérieures

en vue de l’obtention du grade de Maitrise

En Biochimie

“July 2007”

© “Hamsa Dhwani Tadepally” ,2007

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Universitéde Montréal

Direction des bibliothèques

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faculté des études supérieures

Cette thèse intitulée

“Evolution of C2H2-Zinc finger genes in mammalian genomes”

Présentée par:

“Hamsa Dhwani Tadepally”

a été évaluée par un jury composé des personnes suivantes:

“Martine Raymond”

Président-rapporteur

“Muriel Aubry”

Directrice de recherche

“Gertraud Burger”

Co-directrice

“Nicolas Lartillot”

Membre dujmy11

Résumé

Les gènes de doigt de zinc de C2H2/Kruppel (C2H2-ZNF) encodent la plus grande classe

des facteurs de transcription chez Phomme. Ces gènes constituent une des plus grandes

familles de gène chez les mammifères et sont souvent trouvés sous forme de regroupements

de gènes juxtaposés sur les chromosomes. Par une recherche extensive basée sur des

similitudes de séquences visant à d’identifier l’ensemble des gènes C2H2-ZNF du génome

humain, nous avons assemblé un répertoire complet de 718 gènes C2H2-ZNf humains. Les

gènes C2H2-ZNF ont été classifiés en sous-familles en fonction des domaines effecteurs N-

terminaux aux quels ils sont associés. Nous avons constaté que la sous-famille encodant un

domaine KRAB comprend 45% de tous les gènes C2H2-ZNF et est par conséquent fa

plus grande sous-famille de gènes à motifs doigt de zinc. De plus, nous avons identifié 81

regroupements de gènes C2H2-ZNf qui correspondent à 70% de tous les gènes C2H2-

ZNf. Presque 90% des gènes C2H2-ZNF appartenant aux sous-familles KRAB et SCAN

sont trouvés sous forme de regroupements. Pour mieux comprendre l’évolution des gènes

C2H2-ZNF, nous avons par la sUite assemblé un répertoire complet de tous les

regroupements de gènes C2H2-ZNF humains ainsi que de leurs contre-parties dans les

régions synténiques des génomes de chimpanzé, de souris, de rat et de chien. Une analyse

systématique de ce répertoire chez ces mammifères a révélé qu’il existe une variation dans

le nombre de regroupements et de gènes faisant partie de ces regroupements parmi les

primates, les rongeurs et les canins. Cette variation suggère que ces gènes ont évolué de

façon différentielle chez les mammifères. Des études phylogénétiques de plusieurs

regroupements de gènes C2H2-ZNf choisis indiquent qu’outre une duplication‘J’

différentielle, la perte de gènes dans certaines espèces a condujt à des répertoire différents

de gènes C2H2-ZNF chez les mammifères. En plus des variations spécifiques aux espèces

dans le nombre de gènes, nous avons également mis en évidence une variation chez des

orthologues dans le nombre de motifs de doigt de zinc et la présence de domaines

effecteurs, ces derniers étant souvent perdus par dégénération. En conclusion, sur la base

principale de ces résultats et de l’étude de la structure exon-intron des gènes C2H2-ZNF,

nous proposons un nouveau modèle pour lévolution de leurs sous-familles selon lequel les

sous-familles les plus anciennes seraient dans l’ordre SCAN> SCAN-KRAB > KRAB.

iv

Abstract

The C2H2/Kruppel zinc finger genes (C2H2-ZNF) encode the largest ciass of transcription

factors in hurnans. These genes constitute one of the largest gene families in mammals and

are often found in ciusters. Using an extensive similarity search on the hurnan genorne to

identify ail C2H2-ZNF genes, we assembled a comprehensive repertoire of 718 human

C2H2-ZNF genes. The genes were grouped into subfamilies based on the N-terminal

effector domains they were associated with. We found that the KRAB-domain encoding

subfarnily constitutes 45% of the total C2H2-ZNF genes and hence is the largest

subfamiiy of zinc finger genes. In addition to this, we also identified 8 1 C2H2-ZNF clusters

which constitute 70% of the total genes. Almost 90% of the C2H2-ZNF belonging to the

KRAB and SCAN subfamilies were found in ciusters. We then assembled a comprehensive

repertoire of ail the hurnan C2H2-ZNF clusters and their syntenic counterparts in

chimpanzee, mouse, rat and dog genomes. A systernatic analysis of ah the syntenic clusters

reveaÏed a variation in the numbers of clusters and the genes within clusters among

primates, rodents and canines indicating differential pattems of evolution in mammals.

Evolutionary analysis of few selected C2H2-ZNf syntenic clusters in the five mammals

studied suggested that not only differential duplication, but also gene ioss has led to

different repertoires in mammahian genomes. In addition to lineage- and species-specific

variation in the number of genes, we aiso find a variation among orthologs in the number of

zinc finger motifs and in the presence of the effector domains, the later being often lost by

sequence degeneration. finally, based on the above resuits and on the analysis of the exon

intron structure of the various C2H2-ZNF genes, we propose a model for the evolution ofy

their subfarnilies suggesting that the more ancient subfarnilies are in sequential order

SCAN> SCAN-KRAB > KRAB.

Keywords: C2H2/Kruppel, zinc finger, gene farnily, tandem repeats, gene duplication,

gene loss, evolution.

vi

List of abbreviations

DNA: Deoxyribonucleic Acid

RNA: Ribonucleic Acid

BIB: Broad-Cornplex, Tramtrack and Bric-a-bric

POZ: Pox virus and Zinc finger

KRAB: Kmppel Associated Box

SCAN: SRE-ZBP, CTfin5l, AW-l andNumberl8 cDNA

KRI motif: KRAB Interior motif

1g: Immunoglobulin

ZNF45: Zinc finger 45 (protein or gene)

ZNF91: Zinc finger 91 (protein or gene)

BLAST: Basic Local Alignment Search Tool

MUSCLE: Multiple Sequence Comparison by Log-Expectation

OR: Olfactory Receptor

VH and VL domains: Heavy & Light chains ofthe Variable domain oflmmunoglobulin

molecule

KRAB C2H2-ZNF: C2H2-Zinc finger proteins associated with a KRAB domain

SCAN C2H2-ZNF: C2H2-Zinc finger proteins associated with a SCAN dornain

BIB C2H2-ZNF: C2H2-Zinc finger proteins associated with a BTB domain

KAP-1: KRAB associated protein 1

TIF1fl: Transcription Intermediaiy Factor I 3

xiv

List of definitions

Homology: This is a concept that signifies common ancestly.

Orthologs: Genes in different species, which are similar to each other and originated from

a common ancestor, regardless oftheir functions through a speciation event.

Paralogs: Genes that are derived from a duplication event, in the sarne species or different

species. They may or may not have the same function.

Gene duplication: Duplication ofa region ofDNA that contains a gene; it may occur as

an en-or in homologous recombination, a retrotransposition event, or duplication of an

entire chromosome.

Phylogenetic tree: This is also called an evolutionary tree, and shows the evolutionary

interrelationships arnong various species or other entities that are believed to have a

common ancestor.

Synteny: This describes a common order of genes, especially between related species.

xv

Acknowledgements

Questions and Answers are what life at the university seems to be about. Whiietiying to answer the questions about zinc fingers during my thesis, I also seem to haveleamt a lot about myseif These three years at UdeM have been a wonderffil leamingexperience both academically and personally.

First and foremost, I would like to thank rny thesis supervisor, Muriel Aubiy for acceptingand offering me the chance to be her student and work on zinc fingers and for ah theguidance and encouragement. For teaching me that what you leam during the wholeprocess of research is as important as the end resuit. For supporting me when I wasstruggiing with rny courses in French. For ail the days and nights of constant guidance shegave me for the thesis and for ahi the weekends at North Hatley. for giving me theopportunity to go to the SMBE 07, which by far has been the most exciting experience ofmy life. Dr.Muriel, thank you very rnuch foi- eveiything. This experience has made me theconfident person I am today.

Gertraud Burger, my co-supervisor for lier vahuabie guidance and suggestions. Foi- givingme the opportunity to interact with everyone from the Bioinformatics group and supportingme to let me continue in the Masters program.

Franz.B.Lang, Nicolas Lartihlot, Herve Philippe, Henner Brinkrnann and Amy Hauth for ailthe helpful guidance, discussions and constructive comments. Ahian Sun for the assistancewith the hardware and software problems I had.

My labrnates, Patricia, Deiphine, Xavier, Imene, Phuong and Hadrian for ail the help, forbeing so nice and ahways making me feel welcome in the iab.

I would like to thank my friends Uma, Reena and Ekta for supporting me during thedifficuht times I had. Karthik for being my computer guru. My girls Lakshmi, Gayatri,Sujata, Shivani and Ramaa for taking care of me and putting up with me during the difficuittimes of my thesis. Siva for helping me out at the university every tirne I had a probiem.Nagu who always let me take my frustrations and bad rnoods on him and for aiways beingthere to talk. Preethi and Kavitha for just being rny friends.

Last but not the least; this entire experience would be at most an unftilfihled dream were itnot for my loving family. I would like to express my gratitude to rny parents, Dr.NagenderSwamy and Vijaya Lakshmi for supporting my dreams and aspirations, for ietting me takemy own decisions. make mistakes, leam and grow. My sister Vamsee Priya, my brotherCharan for always being there for me and aiways taking care of me no matter what and rnybrother-in-law Sanjay.

xvi

Table of contents

Identification of the Jury ii

Résumé iii

Abstracty

Table of contents vii

List of figures ix

List of Supplementary f igures xi

List of Tables xii

List of Supplementaiy Tables xiii

List ofabbreviations xiv

Acknowledgernents xvi

Chapter 1 iNTRODUCTION

1.1 Transcription factors 2

1.2 The C2H2 zinc finger gene farnily 6

1.2.2 The tandemly organized C2H2 zinc finger motif 7

1.2.3 The N-terminal regulatory dornain of C2H2 zinc finger proteins 9

1.3.Gene farnilies and Gene duplication 15

1.3.1 GeneFamilies 15

1.3.2 Gene Duplication and Gene Loss: Two important evolutionary mechanisms

guiding the evolution of gene families in mammals 1$

1 .4 Infening gene duplication and gene Ioss 25

1 .5 Previous Studies addressing zinc finger gene evoltition 2$vii

1.6 Hypothesis and Objective.30

Chapter 2. ARTICLE 32

Evolution ofC2H2-zinc finger genes in mammals: Species-specific duplication and loss at

the level ofclusters, genes and their frmnctional dornains 33

Chapter 3. DISCUSSION 145

3.1 The C2H2-ZNf genes in the human genome 147

3.2 Variation in the numbers ofC2H2-ZNF genes in mammalian clusters 148

3.3 Evolution of C2H2-ZNF genes in mammals through differential expansion and loss

150

3.4 Evolution ofthe C2H2-ZNf genes through duplication or loss of zinc finger and N

terminal effector motifs 152

3.5 Birth and Death model ofevolution 153

3.7 A few concems to the study 156

3.8 Merits ofthe study 159

3.9 Perspectives 160

REFERENCES 161

viii

List of Figures

INTRODUCTION and DISCUSSION

Figure 1: The basic structural unit ofa C2H2 zinc finger protein 8

Figure 2: The Regulatoiy domains associated with C2H2 Zinc finger proteins 14

Figure 3: Darwin’s evolutionary tree 20

figure 4: Schernatic representation ofspeciation and duplication 22

Figure 5: Schematic representation of different evoÏutionaiy processes shaping the gene

farnilies in different species 24

Figure 6: Inferring gene duplication and loss events from a gene tree in comparison with

the species tree 27

Figure 7: Birth-and-death model ofevolution 154

Figure 8: Plot of the amino acid sequence lengths of ail the C2H2-ZNF in the human

genome 158

ARTICLE

Figure 1: Flowchart of the analysis procedure of C2H2-ZNF genes and clusters 69

Figure 2: Distribution of ail the singletons and clustered genes from the various human

C2H2-ZNF sub-farnilies and gene composition ofthe C2H2-ZNf clusters 70

Figure 3: Differential expansion and loss of C2H2-ZNF clusters in five mammalian

genomes 72

Figure 4: Evolutionary scenarios in the phylogenetic tree 74

ix

Figure 5: Phylogenetic analysis ofC2H2-ZNf genes in cluster 19.12 ofhuman and its

syntenic counterparts in other mammals 76

Figure 6: Physical maps showing the organization of the hurnan C2H2-ZNF from cluster

19.12 localized on 19q13.4 and its syntenically homologous counterparts in other mammals

7$

Figure 7: Variation in the numbers of zinc finger motifs in mammals and in the presence of

consewed N-terminal dornains in orthologs 80

Figure 8: Model for the evolution ofthe SCAN, SCAN-KRAB and KRAB C2H2-ZNF

subfarnilies 83

X

List of Supplementary Figures

ARTICLE

Supplernentaiy Figure 1: Distribution of intergenic distances between 71$ C2H2-ZNF in

the human genome 87

Supplernentary Figure 2: Comparison of the number ofC2H2-ZNF genes in the 40 human

clusters containing at least 3 C2H2-ZNF and their syntenic counterparts in four other

mammals 8$

xi

List of Tables

INTRODUCTION

Table 1: Different types ofDNA binding domains 4

xii

List of Supplementary Tables

ARTICLE

Supplementaiy Table Si: Comprehensive catalogue of the 718 C2H2-ZNf genes in the

human genome 91

Supplernentaiy Table $2: Comprehensive surnmary ofthe organization of ail C2H2-ZNF

found as singletons or in clusters on each human chromosomes and classified with respect

to the various C2H2-ZNF sub-farnilies 112

Supplementary Table $3: Gene organization of the 81 hurnan C2H2-ZNF clusters 113

Supplementary Tabie S4: Comprehensive catalogue of the C2H2-ZNF genes from the 81

human clusters and their syntenic counterparts from other mammalian genomes

(chimpanzee, mouse, rat and dog) 1 15

xiii

Chapter L INTRODUCTION

1.1 Transcription Factors

A veiy important problem in biology is trying to understand the mechanisms by

which particular genes are expressed in a temporal or a tissue-specific manner. The process

through which a DNA sequence is copied by an RNA polymerase enzymatically to produce

compÏementary RNA is called Transcription.

The transcription process in prokaryotes and eukaryotes differs in the fact that an

RNA polyrnerase alone can initiate transcription in prokaryotes. In contrast, eukaryotes

have a much more complex transcriptional regulatory mechanism. In addition to the RNA

polymerase, eukaryotic genes need an initial assernbly of transcription factors at the

promoter (Pabo and Sauer 1992).

Transcription factors are proteins involved in the regulation of gene expression by

binding to the promoter elernents upstream of genes. They are composed mainly of two

functional regions 1) a DNA-binding dornain and 2) an Effector domain.

The DNA-binding dornain consists of amino acids that recognize specific DNA

bases generally near the start of transcription. Based on its structure, the DNA-binding

domain is classified into different types as detailed in Table I.

1. Zinc finger

2. Helix-tum-helix

3. Leucine zipper domain

4. Winged helix

5. ETS domain

2

6. Helix-loop-helix

7. Immunoglobulin fold

In addition to a DNA-binding dornain, transcription factors also contain an effector domain.

This domain often interacts with proteins to either inhibit or activate transcription.

Transcription factors can thus act as transcriptional activators or repressors that control

gene expression by acting directly on the RNA-polymerase-containing complex bound at

proxirnity of the transcription initiation sites and/or on proteins involved in the assembly of

chromatin, the complex of DNA and proteins that make up chromosomes (Roberts 2000).

Transcription factors bring about these changes either by themselves or indirectly by

recruiting co-factors that are called co-repressors or co-activators (Roberts 2000) depending

on their effect on transcription. Co-repressors or co-activators do not bind DNA directly,

but are recruited to the gene by the effector domain of transcription factors.

9

ETS domain: This dornain is 85-90 arnino acidslong. It was discovered in the ETS oncogene.Three aipha-helices and a 4-strand beta sheetfold into a domain. The third helix is therecognition helix.Example:The Elki-E74DNA complex, where Elk-1 is amember of a large group of eukaryotictranscription factors with ETS domain.Alpha helices are in blue and beta-strands inyellowHellx-Loop-Hellx: This motif has two alphahelices connected by a loop. Generallytranscription •factors with this ioop are dimeric.A smailer helix allows dimerization while theother larger helix facilitates DNA binding.Example:Iwo alpha helices (in Red) connected by a loop(in Green) to form a domain.

Immunoglobulin fold: This is also called an ailf3 protein fold, which has a 2-layer sandwich of7 antiparallel f3-strands arranged in two f3-sheets.

Example:Hurnan Tenascin with its immunoglobulin fold,fibronectin type Iii, coloured from Blue (Nterminus) to red (C-terminus).

Winged helix: This motif has 110 aminoacids. Each dornain bas four alpha-helices andtwo beta-sheet strands.

Example: Alpha helices are in purple and betastrands are in yellow.

4

5

Table 1: Different types ofDNA bïnding domaïns

DNA-bindinZinc lingerA zinc finger has two antiparallel 13 strands andan a helix. Two cysteines and two histidinesinteract with a zinc ion to form a finger likestructure.

Example: The two cysteines on the beta-strandin green can be seen interacting with the twohistidines in orange on the aipha-helix. Theinteracting zinc ion is shown in red in the center.

llellx-TurnHeiïx: This is a major structuralunit capable of binding DNA.It has two aipha-helices which are joined by ashort stretch ofamino acids (turn).

Example: Helix-turn-helix (green and yellow)of bacteriophage lambda, which binds to DNA(blue and cyan).

Leucine zipper domain: it consists of a shortalpha helix with a leucine residue at everyseventh position.Example:The Ap-1 dimer formed by Fos and Junhomologous proteins. The leucine zipper motifbas two Œ-helices which look like a zipper withthe leucine residues (in Green) lining the zipper.

4

1.2 The C2H2 zinc finger gene famïly

0f the rnany large families encoding transcription factors that have been identifled,

zinc finger genes of the C2H2 type constitute the largest one (Schuh, Aicher et al. 1986;

Bellefroid, Lecocq et al. 1989). The C2H2 motif encoded in these genes typically includes

two cysteines and two histidines coordinating a zinc ion. This motif was first identified in

the TFIIIA of Xenopus leavis and later in the Krtippel drosophila segmentation gene

(Miller, McLachlan et al. 1985; Schuh, Aicher et al. 1986). Thus, the C2H2 zinc finger

genes are often refeired to as TFIIIA/Kmppel type of zinc finger genes.

Known to constitute one of the ten largest gene families LPfam databasel, these

zinc finger genes are found not oniy in eukaiyotes but also in prokaiyotes. Members of

C2H2 zinc finger family have now been identified in ail kingdoms of life i.e. eubacteria,

archaebacteria, protists, ftingi, animais and plants (Bouhouche, Syvanen et al. 2000;

Moreira and Rodriguez-Valera 2000). Throughout evolution, there bas been a massive

expansion in the numbers of the C2H2 zinc finger genes (Lander, Linton et al. 2001;

Venter, Adams et al. 2001). Noticeably, human beings are predicted to have more than 700

zinc finger genes often found in a ciustered organization (Bellefroid, Lecocq et al. 1989;

Looman, Abrink et ai. 2002).

While most of the C2H2 zinc finger genes characterized have been described as genes

encoding transcription factors which bind to DNA, some are also known to encode RNA

binding proteins that may thus participate in RNA metabolisrn or maturation (Theunissen,

Rudt et ai. 1992; Grondin, Bazinet et al. 1996).

6

1.2.1 Structure of the proteins encoded by C2H2 ZNf genes

The C2H2 zinc finger transcription factors generaÏly consist of two essential regions

1) the C2H2 zinc finger region containing in most instances several zinc finger motifs

organized in tandem and 2) the N-terminal regulatoiy domain

1.2.2 The tandemly organized C2H2 zinc finger motif

The C2H2 zinc finger proteins are composed of zinc finger motifs which form the

zinc finger region of the protein. Each motif is a highly conserved sequence of 28 amino

acids (CX24CX3FX5LX2HX34HTGEKPYX, where X is any amino acid). Each motif is

separated from the following one by a highly conserved linker region (TGEKPYX, where

X is any arnino acid) (Miller, McLachlan et al. 1985; Wolfe, Nekiudova et al. 2000;

Loornan, Abrink et al. 2002). The basic conserved C2H2 zinc finger structural unit includes

two cysteines and two histidines which interact with a zinc ion and are essential for the

proper folding ofthe motif into a finger like structure (See f igure 1) (Looman, Abrink et al.

2002). C2H2 zinc finger proteins are composed of one or more tandemly organized zinc

finger motifs. The number of zinc finger motifs in the protein varies from one to more than

30 in a few cases (Ruiz i Altaba, Peny-O’Keefe et al. 1987).

7

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B

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Figure 1: The basic structural unit of a C2112 zinc linger protein.

(A) The C2H2 zinc finger motif is present in tandem in the protein. Three zinc linger motifs arc

connected by a conserved Iinker region (TGEKPY). The two cysteines and two histidines which

interact with a zinc ion inc]uding die other conserved residues are shown with their single letter

codes. The residues involved in DNA binding are shown in grey.

(B) The three-dimensional structure of a zinc finger binding domain. Two anti-parallel f3-strands

and one Œ-heÏix interact with a zinc ion as shown in the figure.

8

Nuclear magnetic resonance spectroscopy (NMR) was used to determine the three

dimensional structure of the C2H2 zinc finger motif (Lee, Gippert et al. 1989; Omichinski,

Clore et al. 1990). Two beta strands and one alpha helix form an independently folded

domain with a compact globular structure (See figure 1). The zinc ion, that is tetrahedrally

coordinated betwcen two invariant pairs of cysteines and histidines, connects the 3-sheet

and the a-helix. Four amino acids on the surface of the a-helix in the zinc finger motif

make base specific contacts with three to four bases in the major groove of the DNA helix

(frankel, Berg et al. 1987; Panaga, Horvath et al. 1988; Omichinski, Clore et al. 1992;

Krishna, Majumdar et al. 2003). Aithougli the zinc finger domain has been described as

nucleic acid binding domain, not ail the zinc finger motifs are involved in DNA or RNA

binding. For example, in ZBRK1 zinc finger protein, only the first few fingers are involved

DNA binding and ail the others in protein-protein interactions (Zheng, Pan et al. 2000).

1.2.3 The N-terminal regulatory domain of C2112 zinc fingerproteins

In addition to the zinc finger region, C2H2 zinc finger proteins are also associated

with an N-terminal regulatoiy domain (f igure 2), which regulates subcellular localization

and the gene expression by acting as either a repressor or an activator by itself or by

associating with other factors (Collins, Stone et al. 2001).

9

The regulatoiy domains associated with C2H2 Zinc finger proteins are

j. BTB/POZ domain

ii. KRAB domain

iii. SCAN dornain

j. The BTB domain

The BTB domain (Broad-Cornplex, Tramtrack and Bric-a-bric) also known as

the POZ domain (Pox virus and Zinc finger) is a 120 arnino acid conserved dornain found

to be associated with both DNA and actin-binding proteins. The 3TB domain is involved in

protein-protein interactions (Collins, Stone et al. 2001). As a part of DNA binding proteins,

the BTB/POZ domain is known to be a dirnerization domain which, in a few cases also

recmits co-repressors (such as N-CoR, STN3A or SMRT) and acts a repression domain.

When found in association with C2H2 zinc finger transcription factors, the BTB domain is

generally located N-terminal to the zinc finger region. Thus, by mediating oligornerization

and in some instances interaction with co-factors, the BTB domain can lead to chromatin

remodeling and change in gene expression (Melnick, Carlile et al. 2002).

ii. The Kruppel-Associated Box (KRAB) domaïn

Another well known example of an N-terminal regulatory domain associated

with C2H2 zinc finger proteins is the Kruppel-Associated Box or the KRAB domain

(Bellefroid, Poncelet et al. 1991; Rosati, Marino et al. 1991). KRAB domains are almost

10

aiways associated with C2H2 zinc finger proteins. An exception to this scenario is the

SSX family ofproteins. These proteins are associated with a “SSX KRAB dornain” which

is distantly related to the KRAB domain from C2H2 zinc finger proteins ( 49% similar)

but are flot associated with zinc fingers (Collins, Stone et al. 2001; Urrutia 2003).

Unlike the C2H2 zinc finger proteins which are present in organisms ranging

from bacteria to humans, the KRAB dornain as seen in C2H2 zinc finger proteins is present

only in vertebrates, more specifically in tetrapods (Looman, Abrink et al. 2002). However,

a recent study identified a sea urchin homolog to the mammalian Meistez protein which

includes a tandem array of C2H2 zinc finger motifs, a SET dornain and a sequence with

some sirnilarity to the “SSX-KRAB domain” (Birtie and Ponting 2006). This suggests the

presence of the KRAB domain in the common ancestor of echinoderms and vertebrates. A

further study of these proteins in ftingi and plants identified a 26 amino acid motif called

the KRI motif which was found to be similar to the aipha-helical regions of KRAB and

present in ail eukaryotes. This indicated that the KRI motif was present in the last common

ancestor of animais, plants and fungi and is the progenitor of the KRAB dornain.

The KRAB domain is most abundant in mammals (Lander, Linton et al. 2001;

Venter, Adams et al. 2001; Waterston, Lindblad-Toh et al. 2002). For example, about one

third of the mouse C2H2-ZNF are associated with KRAB (Benn, Antoine et al. 1991;

Waterston, Lindblad-Toh et al. 2002). The KRAB domain is mostly associated with more

than 5 C2H2 zinc finger motifs in a protein, justifying the name “Multifingered protein”

(Bellefroid, Poncelet et al. 1991). Many genes encoding the KRAB containing proteins are

found in a clustered organization as opposed to the ones found as singletons (Bellefroid,

11

Marine et al. 1993; Shannon, Kim et al. 199$; Chung, Schafer et al. 2002; Rousseau

Merck, Koczan et al. 2002; Hamilton, Huntley et al. 2003).

The KRAB domain is 75 amino acids long and is divided into two boxes, Box A

(-38 amino acids) and Box B (32 amino acids) (Looman, Abrink et al. 2002; Urrutia

2003). A variant of the B box, called b box also exists. Some C2H2 zinc finger proteins

have another box following the A box, called the C box (21 amino acids). Each of these

boxes is encoded by different exons and separated by introns of vaiying lengths (Loornan,

Heilman et al. 2004). The KRAB domain functions as a potent repressor of transcription

(Margolin, Friedman et al. 1994). The KRAB A box plays an important role in repression

by binding to co-repressors, while the KRAB B box doesn’t have transcriptional activity

but is known to enhance the repression activity of the A box (Witzgall, O’Leary et al.

1994). The process of transcription repression is mediated by KAP-1, also called

transcription intenriediary factor 13 (Tlflt3) which is a co-repressor that interacts with

KRAB A (Friedman, Fredericks et al. 1996; Germain-Desprez, Bazinet et aI. 2003). The

KRAB domain of C2H2 zinc finger proteins recruits the KAP I co-repressor to DNA,

which results in the formation of a heterochromatin like complex and leads to gene

silencing (Pengue, Calabro et al. 1994; Kim, Chen et al. 1996; Moosrnann, Georgiev et al.

1996; Pengue and Lania 1996).

iii. The SCAN domain

The SCAN domain like the KRAB domain is another vertebrate specific domain

only associated with C2H2 zinc finger proteins (Williams, Khachigian et al. 1995; Looman,

12

Abrink et al. 2002). The SCAN domain was estimated to be associated with 10% of the

total C2H2-ZNF present in the human genome (Collins, Stone et al. 2001; Edeistein and

Collins 2005). Also known as the LeR domain because of its leucine rich primaiy

structure, the SCAN domain is named after the four proteins it was initially identifled

(SRE-ZBP, CTfin5l, AW-1 and Numberl8 cDNA) (Urrutia 2003). In addition to being

associated with the C2H2 zinc finger proteins, the SCAN domain containing proteins are

sometimes associated with a KRAB domain having the organization SCAN-KRAB

(C2H2) or in very few cases KRAB-SCAN-KRAB-(C2H2) (Edeistein and Collins 2005;

Huntley, Baggott et al. 2006).

Structural studies on the SCAN domain indicate that it has 84 arnino acids and is

found to have three to five a-helices which are delineated by one or more proline residues.

Proline residues are also present before and after the SCAN domain (Stone, Maki et al.

2002). The SCAN domain is a homo and hetero-dimerization domain mediating protein

protein interactions by self association and formation of heterodimers between SCAN

family members (Sander, Haas et al. 2000; Schumacher, Wang et al. 2000). The importance

of the dimerization for the transcriptional activity of SCAN-C2H2 zinc finger protein lias

flot been clearly established.

13

Regiilatory Dornain Spacer Zinc linger region

_

- flUBA

VTFED5AVYFSQEEWGLLDPAQRNLYRDvLENY

RNLVSL

—FJT------ -

J.

KRAB b

bHQLFJOEDX I sQLEREEKLWMMIxATQRGDS S>’k !.

nU

SCANPDPEIFRQRFRQFCYQETPGPREALSR LRELCHQ

WLRPEVHTKEQILEL LVLEQF LTI LPKELQAWVQ

EIfflPESGEEAVTLLEDLERELDEPGQQV

. LQNPSIWTGLLCKANQMRLAGTLCDVVIMVDSQE

FEFTILiCTSK14FEILFRRNSQHiTLDFLSPK., ., . TFQQILEYAYTATLQAKAEDLDDLLYAAEILEIE

Y LEEQC LKM L

B

Figure 2: The Regulatory domains associated with C2H2 Zinc linger proteins.

(A) The different combinations of dornains associated with zinc finger proteins are shown.

Zinc finger proteins generally have three main regions: The Regulatory domain, the Spacer

and the Zinc finger region. (B) The consensus sequence of the domains KRAB (A, B, b and

C boxes), SCAN and BTB. The residues essential for binding KAPI and thus for repression

are shown in KRAB A underlined in red.

14

1.3 Gene familles and Gene duplication

1.3.1 Gene Families

A gene family colTesponds to a set of genes that are grouped based on their shared

homology, biologicai or biochemical activity, sequence motifs or similarities in stntcture.

Because they consist of a large number of genes, gene families are the most informative

systems to study evolutionary dynamics of genes. Nuclear genomes have many multigene

families and their studies provide dues to the evolutionaiy forces that have shaped these

genomes (Ohta 2000; Thomton and DeSalle 2000). Mammalian genornes in particular have

large numbers of genes organized in gene families (Demuth, Bie et al. 2006). Some gene

families have uniforrn copy numbers of genes in ail species (Thomton and DeSalle 2000),

while there are gene families like the Immunoglobulin gene family, the Olfactory receptor

gene family and the C2H2 zinc finger gene family which have a large variation in the

number of genes across different species . The variation in the gene numbers of these

families and diversity in ftinction, suggests that gene duplication and/or gene ioss have

played an important role in shaping different mammalian genomes.

I. The Olfactory Receptor gene family

Olfactoiy receptor (OR) genes form the largest known multigene family in

mammalian genomes (Glusman, Bahar et al. 2000) and code for membrane receptors that

are responsible for olfaction, the sense ofsmell. OR genes are present in various vertebrates

ranging from lampreys to humans. The OR proteins belong to the G-protein coupÏed

15

receptor family which have seven transmembrane dornains. OR genes are divided into 2

classes based on their protein sequence similarity (Glusman, Bahar et al. 2000; fuchs,

Glusman et al. 2001). 0f the two classes, Class I genes first identified in fish but also found

in mammals are specialized in water-soluble odorants and the Class II genes specialized for

airbome odorants are specific to tetrapods.

The number of the OR genes is quite varied in different genomes. Rodents have

nearly twice as many as the number present in human, chimpanzee or dog (Niimura and

Nei 2005). The Human genome has more than half of the -900 OR genes as pseudogenes.

In contrast, the mouse genome bas l300 OR genes of which only one-fourth are

pseudogenes. $tudies on the human, chimpanzee and mouse OR gene repertoires indicate

that there are species-specific expansion and pseudogenization signifying different

selection pressures in humans, chimpanzees and mouse owing to their different sensoiy

requirements (Sharon, Glusman et al. 1999; Glusman, Yanai et al. 2001; Lapidot, Pilpel et

al. 2001; Gilad, Man et al. 2005; Niimura and Nei 2005). Evolutionary analysis of the

human, mouse and chimpanzee datasets indicate the presence of clustered organization

which is generally well conserved in these genornes. Analyses of the clusters indicate that

there are tandem arrays of the OR genes which appear to have arisen by tandem duplication

and several chromosornal rearrangements. The difference in the numbers of OR genes in

hurnan and mouse has been attributed to gene duplication and loss events (Sharon,

Glusman et al. 1999; Niimura and Nei 2005).

16

ii. The Immunoglobtilin gene family

The immunoglobulin gene family represents an example where its two subfamilies,

the immunoglobulin heavy variable region sub-farnily and immunoglobulin light chain

variable region subfamily, have co-evolved by valying in gene number and extent of

diversity in different species ($itnikova and Sti 1998). An immunoglobulin molecule is a

tetramer with two identical heavy chains and two identical light chains which forrn a Y

shaped structure. Each of these chains has a variable (V) and constant (C) domain. The VH

and VL domains have the complementarity determining regions, called the CDRs which

form the sites of interaction with antigens. Analyses of these two sub-farnilies of genes

from various species of amniotes identified that these gene families have diversified

throughout the course of evolution (Sitnikova and Su 1998). Different coordinated loss and

duplication events have led to different species-specific gene repertoires.

iii. The C2112 zinc finger gene family

In addition to the above mentioned gene families, the C2H2 Zinc finger gene farnily

is another example of a large multigene family with varying number of genes in different

species. Over the course of evolution, this gene famlly bas expanded drastically in

mammalian genornes (e.g. — 400 in mouse and 700 in human) (Venter, Adams et al.

2001; Waterston, Lindblad-Toh et al. 2002). Several studies involving these genes in the

human genome have indicated that tandem duplication events are responsible for the

17

clustered organization of this family (Shannon, Kim et al. 199$; Elemento and Gascuel

2002; Elemento, Gascuel et al. 2002; Tang, Waterman et ai. 2002; Bertrand and Gascuel

2005; Huntley, Baggott et al. 2006). A few instances of evoiutionary studies ofthese genes,

within the human genome and among a few mamnialian genomes document cases of

species-specific duplication (Dehal, Predki et ai. 2001; Shannon, Hamilton et ai. 2003;

Huntley, Baggott et al. 2006).

Ail these examples of gene families suggest variation in number among

different species involving different duplication and ioss events. The gene family size could

vaiy based on the ftinctionai relevance of the gene farniiy in the organism. These examples

also indicate the importance of studying the gene families to give dues on the evolutionaiy

mechanisms which led to different sizes of gene families.

1.3.2 Gene Duplication ami Gene Loss: Two important evolutionary

mechanisms guiding the evolution of gene famïlîes in mammals

Considering the extremely large numbers of genes constituting gene families

(Demuth, Bie et al. 2006), it is interesting to study their organization and the evolutionaiy

mechanisms that created them. A study integrating the information from spatial

organization of the genes with the phylogenetic reiationships between the genes combined

with evolutionaiy information of the species would help provide dues about the evolution

ofthe gene families.

18

In the context of using phylogenetic studies to analyze the evolutionary

reiationships between genes in gene families, one significant term that features in ail studies

is “Romoiogy”. Homology forms the centrai and basic concept of comparative genomics

but is aiso a terni that is often misrepresented and misinterpreted. The terni homology was

introduced by Richard Owen in 1848, where lie defined homology as “the sanie organ

under eveiy variety of form and fttnction”(Francis Darwin 1903) . The importance of

structure and fiinction is ernphasized more in this definition. In an attempt to give an

evoiutionary explanation to hornoiogous structures, Darwin defined homology as “A

structure is sirniiar among reiated organisms because those organisms have ail descended

from a common ancestor that had an equivaient trait” (Darwin 1837) (Figure 3).

When put in the context of molecular sequence comparison, in today’s times,

homoiogy refers in an abstract way to a reiationship which implies a possible common

ancestry and shouid be differentiated from identity2 or similarity3 of sequences. However,

to be substantiated, homology must be confirmed by appropriate phylogenetic studies. It is

important to note that homology does flot say anything about functionai simiiarity

(Thomton and DeSalle 2000))(Fitch 2000).

131Homology: A hypothesis that signifies comnion ancestry between sequences (nucleotide or amino acid)which is prirnarily based on sequence similarity.

2ldentity: The extent to which two (nucleotide or amino acid) sequences are invariant.

Similarity: The extent to which nucleotide or protein sequences are related. The extent ofsirnilaritybetween two sequences can be based on percent sequence identity and!or conservation

19

Figure 3: Darwin’s evolutionary tree.

The figure is Charles Darwin’s first ever sketch of an evolutionary tree from bis book titled

“First Notebook on Transmutation of Species (1837)”.

20

There are three major types of homology in a phylogenetic context which are

orthology, paralogy and xenology. Orthotogy as described by f itch in 1970 is the

relationship between two genes in two different species which originated from a common

ancestor. Two homologous sequences are considered to be “orthologous” if a speciation

event separates them. In contrast, Paralogy signifies the relationship between two genes

which have been formed by a gene duplication event. XenoÏogy, another type of homology

relationship describes the relationship between two genes which have been transferred

between two species by horizontal gene transfer.

Studying the homologous relationships of genes within and between various

genomes and differentiating between orthologs and paralogs is a central aspect of

comparative genomics. figure 4 shows a very simple explanation of the difference between

orthologs and paralogs. The genes Ai, Bi and 32 have evolved from an ancestral gene by

speciation followed by a duplication event in species B. Gene Al from species A is an

ortholog of gene Bi and gene B2 in species B illustrating that one gene in a particular

species may have more than one ortholog in the other. Gene Bi and gene B2 in species B,

which were formed by gene duplication, are paralogs to each other.

21

Speciahon

DRptkWiJrn

Figure 4: Schematic representation of speciation and duplication

Genes Ai, Bi and B2 are formed from an ancestral gene by a speciation and duplication

event. Gene Al from species A has two orthologs in species B, genes 31 and B2. Bi and

32 are paralogs.

22

figure 5 depicts different evolutionary scenarios that one encounters while

studying the evolution of genes in gene families are depicted to explain the relationships

between genes in ternis of orthologs, paralogs and gene Ioss. An ancestral gene undergoes

duplication in species O to give the genes, A, B and C. This is followed by a speciation

event with genes Ai, Bi and Clin Species i and genes A2 and B2 in Species 2. The gene

Al is an ortholog of A2 and Bi is an ortholog of 32. The gene Cl does not have a

corresponding ortholog, as the Species 2 lost the gene after speciation. The genes AI, Bi

and Cl are paralogs within species 1 and, A2 and B2 are paraiogs within species 2.

Furthermore, as explicitly pointed out recently by fitch (Fitch 2000) and as often ignored,

gene Ai (species i) is also a paralog of gene B2 (species 2), gene Bi (species 1) is a

paraiog of gene A2 (species 2) and gene Cl is paralog of gene A2 and B2 (species 2). f rom

these explanations, it is clear that orthologs are homologous genes residing in different

species, while paralogs may not only refer to the homoÏogy relationship between genes

from the same species but also from different species. It is essential to understand that both

orthologs and paralogs are free to diverge and do not necessarily aiways have the same

function (Thornton and DeSalle 2000).

23

Duplk&dsn ewm

Geiz, Ie

Figure 5: Schematic representation of different evolutionary processes shaping the

gene familles in different species.

Gene duplication, speciation and loss lead to the formation of genes Ai, Bi, Cl in species

1 and A2 and B2 in species 2.

csO

1Spci1in ewiu

24

1.4 Inferring gene duplication and gene loss

That two genes are homologous is a hypothesis that needs to be studied and

analyzed to be able to derive the relationships between the genes to be either orthologs or

paralogs. Studying and analyzing the relationships between gene farnilies i.e. evaluation of

orthology or paralogy requires a well formulated approach. In order to be able to postulate

theories on how related genes evolved from an ancestral gene i.e. by gene duplication, gene

loss or by speciation, one needs to assess homology relationships using a well founded

phylogeny.

The first step in assessing homology is a sequence alignment of the molecular

sequences be it nucleotides or arnino acids. This gives a preliminary measure of possible

homology which can then be assessed using a phylogeny. A welI supported phylogeny

gives the evolutionaiy relationships bePveen the genes in relation to one another.

Comparison of a gene phylogeny between genes with the taxonomic relationships between

species, allows gene duplication and loss events to be assessed and roughly dated. As an

example, figure 6 shows the different scenarios of gene duplication and gene Ioss. In

figure 6A, it can be seen that a duplication event prior to the speciation event resulted in

species 2, 3 and 4 created the paralogous gene groups A, B and eventually C. In a

hypothetical situation, suppose the genes 2A, 3C and 4B are missing from the gene tree.

Assuming that the studied sequences were derived from completely sequenced genomes,

the missing genes could either be due to a loss of genes or their possible pseudogenization

in the respective species. That the gene duplication occurred prior to speciation can stili be

25

resolved by superimposing the gene tree with the species tree. Reconciliation between the

species and the gene tree will help resolve the absence of genes 2A, 3C and 4B as can be

seen in figure 6B. This kind of srndy can hence be used to infer the evolution of genes

belonging to gene families within and among species in a phylogenetic context.

26

lA2A3A

4A3C4C4D2B3134E lA

3A

4A3C4C4D2E3B413

Figure 6: Inferring gene duplication and loss events from a gene tree in comparison

with the species tree.

(A) A gene tree showing the phylogeny between genes belonging to species 1, 2, 3 and 4. Genes are

represented as A, B, C and D. A gene duplication represented as x occurred prior to the speciation

event leading to Species 2, 3 and 4.

(B) A species tree showing the relation between species 1, 2, 3 and 4.

(C) A hypothetical situation where the genes 2A, 3C and 4B are missing from the tree as shown in

red. Reconciliation of the phylogenetic tree from (A) with the species tree from (B) helps identify

the flot only the duplication event but also the missing genes to be able to infer loss. Adapted from

(Thornton and De$alÏe 2000)

j

4B

27

1.5 Previous Studïes addressing zinc finger gene evolution

About 2000 of the 30,000 genes in the human genorne code for transcription

factors (Venter, Adams et al. 2001). C2H2-ZNF are the most common of ail the eukaiyotic

transcription factors present in the human genome (encoded by 700 genes). Owing to

these facts, the C2H2 zinc finger gene famiiy has been considered to be an evoiutionary

piayground for genes to develop and differentiate and hence is an interesting famiiy to

study (Looman, Abrink et al. 2002).

The studies pertaining to C2H2-ZNF have mostly been restricted to those associated

with a KRAB domain and more specifically to the human genome. A recent study

identified 423 KRAB C2H2-ZNF ioci organized into 65 ciusters on the human genome

(Huntley, Baggott et al. 2006). Evolutionary studies involving these KRAB C2H2-ZNF

genes indicated that the evolutionaiy reiatedness within and among ciusters was flot only

associated with physical proximity evoiving through tandem duplications but aiso through

distributed duplication and postduplication rearrangement events, which have lcd to the

drarnatic increase in the gene numbers of this famiiy in hurnans (Hamiiton, Huntley et ai.

2003; Hamiiton, Huntley et al. 2006; Huntley, Baggott et ai. 2006). Though present in

clusters, the KRAB C2H2-ZNF are not co-regulated and they show different pattems of

expression (Huntley, Baggott et al. 2006).

2$

A study of one KRAB C2H2-ZNF gene cluster on hurnan chromosome 19,

suggested an evolutionary model showing the presence of certain beta-satellite repeat

structures symrnetrically ordered with the zinc finger genes in the cluster which have

coevolved with the cluster accommodating the expansion of the genes within this cluster

(Eichler, Hoffman et al. 1998).

A statistical analysis using phylogenetic models on four hurnan C2H2-ZNF clusters

on chromosome 19 indicated that positive selection is the driving force involved in the

diversification of the KRAB C2H2 zinc linger genes (Schmidt and Durrett 2004).

Not much is known about the evolutionary histories of these genes in different

mammalian genomes and very few studies have been carried out to comparatively analyze

their evolution. A preliminaiy report on species-specific expansion of these genes, resulted

from one study on a C2H2-ZNF cluster on human chromosome 19 and its syntenically

homologous cluster on mouse chromosome 7 (Shannon, Hamilton et al. 2003) . A study on

the evolution of members of the primate-specific ZNF9 1 KRAB subfamily, which are

mainly found in a chromosome 19 cluster, revealed that this gene subfamily evolved before

the spiit of humans and apes. But afier the split, these genes have continued to evolve

differentially be it through tandem duplications or segmental duplications, leading to

species-specific genes (Dehal, Predki et al. 2001; Hamilton, Huntley et al. 2006).

Inspite of several studies dealing with these genes, there has neyer been a

comprehensive study on the C2H2-ZNF genes and their evolution or their functions. In

29

order to systematically define and analyze the extent of species-specffic duplication and

the role of gene loss in the evolution of these genes, it is important to conduct a

comprehensive study of these gene clusters in mammalian genomes to obtain dues on their

evolution and their possible implications on ffinctions specific to each species.

1.6 Hypothesis and Objective

Previous studies on zinc finger genes have provided evidence that zinc finger

genes have undergone a huge expansion in vertebrate genomes, with a specific increase in

humans. Studies have shown that these genes have been subjected to expansion through

tandem duplication and also of the existence of species-specific duplication events

(Shannon, Kim et al. 199$; Shannon, Hamilton et al. 2003; Harnilton, Huntley et al. 2006;

Huntley, Baggott et al. 2006). A contribution of gene loss in the evolution of C2H2 zinc

finger genes has been suggested but neyer tested rigorously.

The main objective of this thesis is to systematically determine to what extent zinc

finger genes are subrnitted to species-specific expansion and to assess the potential

contribution of gene loss in the evolution of this gene famiiy in mammals. To this end, we

have:

1. Assembled a curated database of ail C2H2-ZNF genes in the hurnan genome and

identify ail the C2H2-ZNF clusters in the human genome

30

2. Searched for syntenically homologous clusters in other cornpletely sequenced

mammalian genomes, narnely chimpanzee, mouse, rat and dog genomes.

3. Perforrned a phylogenetic analysis of C2H2-ZNF genes from the syntenically

homologous clusters.

4. Perfornied a reconciliation of both phylogenetic analyses and physical maps of the

clusters with the species tree accounting for the evolutionary history of the species

in order to infer gene loss and gain.

These studies should allow us to determine the nature of evolutionary events that

shaped this large gene farnily in mammals. In particular, this study wiIl help us to better

infer orthology in the various mammals and better understand the evolution and

relationships between the different C2H2-ZNF subfarnilies.

31

Chapter 2. ARTICLE

32

Evolution of C2H2-zinc finger genes in mammals:

Species-specific duplication and loss at the level of

clusters, genes and their functional domains.

Hamsa Dhwani Tadepalfy, Gertraud Burger and Muriel Aubry*

Department of Biochemistry, Université de Montreal, C.P.612$,

Succ.Centre-Ville, Montreal, QC, H3C 3J7, Canada

To whom correspondence and reprints should be addressed:

Muriel Aubry, Ph.D.

Departrnent of Biochemistiy


C.P. 6128, Succ. Centre-Ville

Montréal, H3C 3J7

Canada

Key words: C2H2/Kruppel, zinc finger, gene family, tandem repeats, gene duplication,

gene loss, evolution.

33

ABSTRACT

C2H2 zinc finger genes (C2H2-ZNF) constitute the iargest class of transcription factors in

hurnans and one of the largest gene families in mammals. Often arranged in clusters in the

genome, these genes are thought to have undergone a massive expansion in vertebrates by a

process involving tandem duplication. However, this view is based on lirnited datasets

restricted to single chromosome or a specific subfamiiy of C2H2-ZNF genes. Here, we

present the first comprehensive study of the dynamic evoiution of the C2H2-ZNF family in

mammals. We assernbled the complete repertoire of hurnan C2H2-ZNF genes (718 in

total), about 70 % of which are organized into 81 ciusters across ail chromosomes. Based

on an analysis of their N-terminal effector domains, we identified

SET- and HOMEO dornain-encoding C2H2-ZNF genes as members of two new C2H2-

ZNf subfamiiies. We searched for the syntenic counterparts of human clusters in other

mammals for which compiete gene data are avaiiable: chirnpanzee, mouse, rat and dog.

Cross-species comparisons show a large variation in the numbers of C2H2-ZNF genes

within homologous mammalian clusters stiggesting differential pattems of evolution.

Phylogenetic anaiysis of selected C2H2-ZNF clusters reveals that differences in C2H2-ZNF

gene repertoires across mammais not only originate from differentiai gene duplication but

also gene loss. Further, we find variations among orthologs in the number of zinc finger

motifs and association of the effector dornains, the later often undergoing sequence

degeneration. Based on these resuits and an anaiysis of the exon-intron organization of

genes from the large SCAN and KRAB domains-containing subfamilies, we propose a new

model for the evolution ofthese subfamilies.

34

This manuscript includes two supplementaiy Figures and four supplementary Tables

INTRODUCTION

The human genome sequence uncovered a large number of gene families oflen

arranged in a clustered organization (Ohta 2000; Thornton and DeSalle 2000; Venter,

Adams et ai. 2001). C2H2 zinc finger (C2H2-ZNf) genes make tip 2 % of ail the human

genes and represent the second largest gene family in humans after the odorant receptor

farnily (Lander, Linton et al. 2001) (Schuh, Aicher et al. 1986; Bellefroid, Lecocq et al.

1989; Messina, Glasscock et al. 2004). The first identified members of the C2H2-ZNF

family are Xen opus TFIIIA and Drosophila Kruppel and thus genes of this family are often

called zinc finger genes of the TFIIIA or Kruppel type (Miller, McLachlan et ai. 1985;

Schuh, Aicher et al. 1986).

Most of the characterized C2H2-ZNF genes code for transcription factors which

bind DNA through their zinc finger region; others bind RNA and their exact function is yet

unknown (Theunissen, Rudt et al. 1992; Grondin, Bazinet et al. 1996). The zinc finger

region is cornposed of a basic structural unit of 28 amino acids (CX21CX3FX5LX2HX3

4HTGEKPYX, where X is any arnino acid), called the zinc finger motif, that is often

repeated in tandem. The two cysteines and two histidines in this motif interact with a zinc

ion, stabilizing the proper folding of this motif (Klug and Rhodes 1987; Lee, Gippert et al.

1989; Rhodes and Klug 1993). C2H2-ZNF proteins often contain an effector domain

aiways located N-terminal to the zinc finger region, such as the KRAB (Kntppel

Associated-Box), SCAN (SRE-ZBP, CTfin5l, AW-1 and Numberl8 cDNA) and BTB

(Broad-Complex, Tramtrack and Bric-a-bric) domains. The first two domains are

35

vertebrate-specific (BelÎefroid, Poncelet et al. 199f; Rosati, Marino et al. 1991; Collins,

Stone et al. 2001), while BTB is also present in insects. The KRAB domain includes the

box KRAB A (—38 amino acids) involved in transcriptional repression and often a second

box, usually KRAB B (—32 amino acids) or in few cases KRAB b or KRAB C (—21 amino

acids) box (Witzgall, O’Leary et al. 1994; Looman, Abrink et al. 2002; Urrutia 2003;

Looman, Heilman et al. 2004). The KRAB A box and the second KRAB B, b or C box are

encoded by separate exons, which are alternatively spliced. The SCAN, also called the

leucine-rich (LeR) domain (— 84 amino acids) (Stone, Maki et al. 2002) mediates protein

protein interactions through dimerization (Sander, Haas et al. 2000; Schumacher, Wang et

al. 2000). The BTB dornain Q—- 120 amino acids) is a dimerization domain that also acts as a

repression dornain in some cases (Melnick, Carlile et al. 2002). In contrast to the SCAN

and KRAB domains which are only present in C2H2-ZNf proteins, the BTB domain is

also found as a part of actin-binding proteins (Coïlins, Stone et al. 2001). C2H2-ZNF

proteins are grouped into different subfamilies based on the type of N-terminal effector

dornain present.

Initial studies on the C2H2-ZNF gene farnily focused on hurnan chromosome 19,

which is particularly enriched in clusters of these genes (Bellefroid, Marine et al. 1993;

Eichler, Hoffman et al. 1998). More recent studies deait more specifically with the KRAB

subfarnily (Mark, Abrink et aI. 1999; Looman, Abrink et al. 2002; Shannon, Harnilton et al.

2003; Huntley, Baggott et al. 2006). The current view is that C2H2-ZNf genes have

undergone a massive expansion during vertebrate evolution by a process involving tandem

duplication (Dehal, Predki et al. 2001; Looman, Abrink et al. 2002; Hamilton, Huntley et

36

al. 2003; Shannon, Harnilton et al. 2003; Harnilton, Huntley et ai. 2006; Huntley, Baggott

et al. 2006). Yet, this view may be biased because it is extrapolated from smali subsets of

C2H2-ZNF genes.

In this report, we reconstructed a global picture of the evolution of the C2H2-ZNF

gene repertoires during mammalian speciation, based on a comprehensive catalogue of ail

human C2H2-ZNf genes and their syntenic counterparts present in clusters in other

mammals. Our study clearly dernonstrates that this gene farnily expanded and contracted

flot only in hurnan but across mammals and in a lineage-specific fashion. In addition, we

discovered evolutionary change of individual C2H2-ZNF orthologs invoiving both

differential duplication of zinc finger motifs and loss of N-terminai effector dornains.

$peciation of mammals is characterized by divergent evolutionary trends at the level of

individual C2H2-ZNF genes as well as the entire farnily. This led us to propose a model for

the evolution of SCAN, SCAN-KRAB and KRAB subfamilies and points to the importance

of comparing complete repertoires rather than C2H2-ZNF genes from specific subfarnilies

for gaining insights into the possible orthologous relationships between genes from varions

genornes.

37

METHODS

Collection of human C2H2 zinc finger genes

We conducted an extensive sirnilarity search to identify the compiete repertoire of

C2H2-ZNF genes in the hurnan genome (assembly NCBI 36). First, we identified ail the

genes annotated as C2H2 and/or Kmppei zinc finger genes by performing an initiai text

term search via Entrez (www.ncbi.nlm.nih.gov). Second, we used PROSITE

(http://www.expasy.com) to identify ah the proteins which had a zinc finger motif of the

C2H2 type as weil as the N-terminal effector domain, if present.

from these searches, the genomic coordinates, chromosome number, position on the

chromosome, number of fingers and identified domains were collected for each of the gene

and protein sequences (initial dataset). A TBLASN (e-vaiue ctttoff le-3) (Gertz, Yu et ai.

2006) search was done against the genome using each of the gene sequences from the

initial dataset as a query. The blast hits were used to generate the final dataset of ah the

identified C2H2-ZNF genes (Suppiementary Table Si).

Identification of C2H2-ZNF gene clusters in the human genome

We anaiyzed the relative positions of C2H2-ZNF genes in the human genorne in

order to identify the C2H2-ZNF clusters. A distribution of the distances between

neighboring C2H2-ZNF genes in the human genome is presented in Supplernentaiy Figure

Si. Two consecutive C2H2-ZNF genes are said to beiong to a ciuster if the distance

between them is 500 kb regardiess of the presence of other genes within the ciuster , a

38

threshold classically used in gene farnily studies (Niimura and Nei 2003). Clusters were

determined for each hurnan chromosome.

Identification of mammalian C2H2-ZNF ciusters syntenically homologous to human

clusters

We searched for clusters hornologous to the human C2H2-ZNf clusters (i.e.

syntenically homologous clusters) in other mammals for which complete genorne

sequences are available. The assemblies used for Pan troglodytes), Mus muscutus, Rattus

norvegicus and Canis famitiaris were chimpanzee Pan Tro- 2.1, mouse NCBI m36, rat

RGSC 3.4 and dog Can fam 2.0. We used the linkage maps of Ensembi

(http://www.ensembl.org); assignment of syntenic clusters is based on the genes flanking

each human cluster and which were mapped in ail the species. Four flanking genes at each

extremity were mapped in most instances. Then, we conducted TBLASTN analysis of the

syntenic regions comprised between the flanking genes, using as queries the amino acid

sequence of the zinc finger region from ail the known human C2H2-ZNF genes from the

corresponding region. A hit with e-value le-4 confirrned the respective hornologous

clusters in the five mammalian genornes. A comprehensive catalogue of the hurnan C2H2-

ZNf clusters and their syntenic counterparts in other mammals is cornpiled in

Supplementary Table S4.

39

Phylogenetic analysis

Phylogenetic analysis was conducted using the amino acid sequences of the zinc

finger region (identified using PROSITE) of C2H2-ZNF genes from selected human

clusters and their syntenically homologous clusters in chimpanzee, mouse, rat and dog. A

multiple sequence alignment of the zinc finger regions of the C2H2-ZNF genes was

generated using the program MUSCLE (Edgar 2004). The alignments were edited to

remove gaps using the program GBLOCKS (Castresana 2000). Maximum Likelihood (ML)

and Bayesian Inference (BI) methods were used to infer the phylogenetic trees and estimate

the clade support. for ML analysis, the program RAxML (RAxML-VI-HPC Version 2.2.1)

(Stamatakis, Ludwig et al. 2005) employing the WAG model of amino acid substitution

was used to reconstruct the best tree. Bootstrapping of 100 datasets was irnplemented. The

posterior probabilities were deterrnined by a Bayesian MCMC method implemented in the

program Mr.Bayes v.3. 1 (Huelsenbeck and Ronquist 2001) to test the robustness of the

topology of the tree infened through ML. One million generations were rcin and the trees

were sampled after every 10 generations.

To determine appropriate outgroups for our analysis, we searched the nr database to look

for close homologs in non-mammals using TBLASTN (e-value eut off le-4). In addition to

the Xfin sequence from Xen opus Ïaevis, we obtained a set of zinc finger genes from

Chicken (Gallus gaïlus, Assembly WASHUC2) specifically selected for each human

C2H2-ZNF cluster based on an extensive similarity search. To select the chicken outgroup,

a TBLASTN (e-value cutoff le-4) search was done against the chicken genome using each

ofthe human C2H2-ZNF sequences derived from the selected cluster of interest as a query.

40

The top 10 hits for each query sequence were ail analysed using a CD-HIT anaiysis

(Identity threshold = 100%, 95% and 90%) (Li, Jaroszewski et aI. 2001) to produce a final

set of non-redundant representative chicken sequences, ail used as a part ofthe outgroups.

Sequence analysis to confirm the Ioss of domains

In the case where loss of a dornain was suspected, we conducted an extensive

sequence analysis to mie out the possibility that these domains would have been rnissed

either due to a frame-shift or inadequate exon-intron spiicing of the gene and thus

inappropriate amino acid translation, preventing recognition by PROSITE

(http://www.expasy.com). Firstiy, for each particular C2H2-ZNF genes where loss of an

N-tennina1 dornain was suggested, we systematically collected the nucleotide sequence of

the region ranging from the stop of translation of the previous gene to the start of

translation of the next gene. We conducted a TBLASTN search of this region using die

amino acid sequence of the dornain of interest (present in the colTesponding orthoiogs and

the consensus of the domains selected from randomly seiected sequences) as a query to

confirm the absence of the domain in the C2H2-ZNF gene of interest. Secondly, we

obtained the exon-intron structure of these genes using the Ensernbl Genorne Browser

(http://www.ensembi.org). In order to search for exonic 01. intronic sequences which may

exhibit significant identity with the nucleotide sequence of the domain of interest. For this

purpose we conducted a BLAST anaiysis of the individual exon and intron sequences with

the nucleotide sequence of the various domains that are present in the coiresponding

orthoiogs.

41

Flowchart of the study

figure 1 summarizes the flowchart of our analysis procedure of C2H2-ZNF genes and

clusters in mammals.

42

RESULTS

Compilation of a comprehensive catalogue of human C2H2-ZNF genes

Previous studies reported the existence of at least 564 C2H2-ZNF genes in the

human genome and suggested that this family may include approximately 700-800 genes

(Bellefroid, Lecocq et al. 1989; Bellefroid, Poncelet et al. 1991). As a first step to study the

evolution of C2H2-ZNF genes, we established a comprehensive catalogue of the C2H2-

ZNf genes in the hurnan genome. By conducting an extensive simiiarity search (see

Methods), we identified 71$ C2H2- ZNF genes (compiied in Supplementary Table Si). 0f

the 718 genes, 66 are annotated as pseudogenes in Genflank. For ail genes, we determined

their exact position on the chromosomes, their orientation, the number of finger motifs and

the effector domains.

These genes are distributed across ail chromosomes of the human genorne

(Supplementaiy Table $2). As reported earlier, chromosome 19 has the highest number

(Venter, Adams et al. 2001) and density ofC2H2-ZNF genes, inciuding 40% (289) ofthe

71$ human C2H2-ZNF genes, whereas this chromosome corresponds to only 2.1 % of the

human genome. More than haÏf (58%) of the C2H2-ZNf genes encode conserved N

tenuinal domains, the KRAB, SCAN and BTB dornains (figure 2A). typically involved in

transcriptional regulation (Kim, Chen et al. 1996; Collins, Stone et al. 2001) and form

different C2H2-ZNF subfamilies. Further, we discovered two additïonal dornains typical of

transcription regulators, the SET and HOMEO domains that are also encoded by C2H2-

ZNf genes. While the KRAB subfarnily represents almost haif of the C2H2-ZNF genes

(45%), SET and HOMEO C2H2-ZNF genes together with members of ail the other

43

subfamilies account for oniy a small percentage (—12%) of the C2H2-ZNF genes (figure

2A).

Clustered organization of human C2112-ZNf genes

It was reported earlier that chromosome 19 is particularly rich in tandemly

duplicated C2H2-ZNf genes and that KRAB C2H2-ZNF genes. are clustered on several

other chromosomes (Dehal, Predki et al. 2001; Rousseau-Merck, Koczan et al. 2002). In

order to trace the duplication history of the entire C2H2-ZNF repertoire, we studied the

distribution of these genes across the whole human genome. Two consecutive C2H2-ZNf

genes were considered to belong to a cluster if the distance between them is 500 Kb,

regardless of the presence of other genes or pseudogenes within the cluster (see Methods).

Using this definition, we identified 81 human C2H2-ZNF clusters accounting for 72 % of

the total number ofC2H2-ZNF genes (518 of the 718) (Supplementary Tables S2 and S3).

The rernaining genes are dispersed as singletons. Among these clusters, 3 1 ¾ include

exclusively tandemly organized C2H2-ZNF genes with no other intervening genes (figure

2B, Supplementary Table S3). The number of C2H2-ZNF genes per cluster ranges from 2

to 76 with an average of 6. As illustrated in the Figure 2B, about 75 % ofthe total number

of C2H2-ZNf clusters has between two to six genes. Consistent with previous reports,

chromosome 19 flot only has the Iargest number of C2H2-ZNF clusters (Supplernentaiy

Table S2) but also hosts the largest clusters (>12 genes) (see figure 2B and

Supplementary Table S3).

44

We find that the large majority of KRAB (89 %) and SCAN (90 %) types of C2H2-ZNF

genes are arranged in clusters (Figttre 2A and Supplementary Table S2). This contrasts with

the BTB subfarnily of C2H2-ZNF genes or those lacking regulatory domains which occur

more offen as singletons in the genome. An analysis of the composition of individual

clusters revealed that two-third of the clusters contains a mixture of various C2H2-ZNF

subfamilies (‘mixed clusters’, Supplementaiy Table S3). The few ciusters made up of a

single C2H2-ZNF gene subfamily (‘pure ciusters’) are ofsmall size (<4 genes).

Identification and comparison of syntenic C2H2-ZNF clusters across mammals

With the ultimate goal to study the evolution of zinc finger genes, we identified and

compiied clusters in completely sequenced mammalian genomes (i.e. chimpanzee, mouse,

rat and dog) that are syntenically homologous to those of hurnan. SyntenicaÏly homologous

clusters were identified by the genes flanking each ciuster. Then, ail the C2H2-ZNF genes

found within the delimited syntenic regions were identified using a TBLASTN search (sec

Methods). The $1 human C2H2-ZNF clusters and their syntenic counterparts in other

mammais are listed in Supplementaiy Table 54, which also inciudes information on the

orientation of the genes in the clusters, their associated domains, the number of zinc finger

motifs and the flanking genes.

Primates (Homo sapiens and Pan troglodytes) stood out for their large number of

both C2H2-ZNF clusters and genes within them, as compared to rodents (Mts musculus

45

and Rattus norvegicus) and Canis fiunitiaris (Figure 3A). The most parsimonious

explanation is that a large expansion of C2H2-ZNF genes occurred in primates, and more

particularly in human (51$ genes in human versus 397 in chirnpanzee) after divergence

from rodents and canines. Rat lias siightly less C2H2-ZNF genes than dog (7%), but 25%

less than mouse. Considering the evolutionary relationship of the species (Figure 3A), these

data suggest that flot oniy species-specific duplication events, as reported earlier (Dehal,

Predki et al. 2001; Hamilton, Huntiey et al. 2003; Shannon, Hamilton et al. 2003), but aiso

loss of family members (suggested here in rodents) may have occurred during the evolution

of mammals. Differential species-specific expansion was reported previously for a subset of

genes from the human ZNF45 subfamily on chromosome 19 compared with its mouse

counterpart (Shannon, Harnilton et al. 2003). Furthermore, expansion of the human KRAB

C2H2-ZNF subfamily was also shown earlier based on draft versions of the genornes of

chimpanzee, mouse and dog (Huntley, Baggott et ai. 2006). However, evidence of C2H2-

ZNF gene or ciuster ioss couid not be definitively obtained in these studies as it required

detaiied analysis of more than two compieteiy sequenced genomes.

Comparing individual syntenic clusters in the mammalian genomes

To distinguish whether differences in the number of C2H2-ZNF clusters are due to

species-specific gene gain or ioss, we systematicaily compared individual syntenic clusters

in the five mammalian genomes studied. The resuits of this analysis point to a differential

evolutionaiy history in mammals. About 60 ¾ ofthe human clusters (49) have syntenicaliy

homologous counterparts in ail the species studied indicating that these C2H2-ZNf clusters

46

predate the divergence of dog, rodents and primates (Supplementary Table S4 and

$upplementary Figure S2). In addition, we found (j) primate specific clusters (14 including

2 human specific clusters), (ii) clusters, present in primates and dog, that were lost in

rodents (8 ciusters including 3 present in mouse but absent in rat) and (iii) clusters present

in primates and rodents but absent in dog (10 clusters) (examples in Figure 3B). Essentially

ail the primate clusters have iarger number of genes than rodent or dog clusters which

reflects a global primate-specific expansion of C2H2-ZNF (Supplementary Figure S2).

Further, in 40% of ail primate clusters, those from human contain more C2H2-ZNF genes

than those from chimpanzee. This indicates that most of the evolutionaiy changes

(duplication and/or loss) occurred late in the primate branch. A similar patteni was seen in

rodents, where almost ail mouse C2H2-ZNF clusters exhibit more genes than their syntenic

rat clusters. While these resuits illustrate that the C2H2-ZNF gene family is rapidly and

independentïy evolving within different Ïineages, insights into the role of gene duplication

and loss in the histoiy of this gene family required rigorous phylogenetic analysis.

Phylogeny of C2H2-ZNF clusters in mammalian genomes

For addressing the relative contribution of gene duplication and loss in the evolution

of C2H2-ZNF genes in mammals, we focused our smdy on selected large human C2H2-

ZNf clusters and their syntenic counterparts in four other mammals. We expected that

larger ciusters would be more informative and possibly more representative of the whoie

genomes. Because of the clarity of evolutionary scenarios observed in the tree, we present

here a detailed phylogenetic analysis of the second largest hurnan C2H2-ZNF cluster (43

47

genes) located on chromosome 19q13.4, that we narned cluster 19.12, and of its syntenic

clusters (Supplementary Table $3 and S4) in other species. For phylogenetic analysis, we

used the predicted amino acid sequences of the zinc finger regions. Genes annotated as

pseudogenes in Genbank or genes containing less than three zinc finger motifs were not

considered in the phylogenetic analysis (noise is expected to be too high if sequences of

only 56 amino acids corresponding to 2 fingers motifs or less were included). Our total

data set of C2H2-ZNF sequences from the hurnan cluster 19.12 and their syntenic

homologs in chimpanzee, mouse, rat and dog consists of 101 protein sequences, including

the outgroup sequences from Xen opus and Chicken. We constructed a phylogenetic tree

using Maximum Likelihood and Rayesian methods. We subdivided the tree into three

groups (figure 5) based on the kind of evolutionaiy scenarios observed i.e. one-to-one and

one-to-many orthologous relationships between genes as weIl as gene loss as defined in

f igure 4. The number of C2H2-ZNF sequences from each species is highlïghted for each

group. Two of these groups are monophyletic with significant (95%) support in both the

Maximum Likelihood and Bayesian analysis (Group I and III).

A detailed analysis of the tree revealed four clades that underwent species-specific

expansion, and two clades, with gene loss in some species. For example, a dog-specific

expansion is seen in the monophyletic Group I, which includes three clustering genes from

human (hZNF331), chimpanzee (pZNF331) and dog (cZNF33Y) which in tum grouped

within a larger clade containing fine additional C2H2-ZNF genes from dog. In addition,

this clade indicates a loss in rodents, due to the absence of mouse or rat genes. Group I

alone illustrates how both species-specific dtiplication in dog and loss in rodents can

48

account for the higher number of genes seen in dog C2H2-ZNf clusters as cornpared to

rodents.

Group II shows more pronounced expansion in hurnan as seen in several clusters.

for example, one of the primate-specific clades includes 17 human genes and 7 chimpanzee

C2H2-ZNF genes (f igtire 5). 0f the 17 hurnan genes present in the clade, only 6 genes

show a one-to-one orthologous pairing with chimpanzee genes. Another well supported

clade includes a single human gene (hZNf677) clustered with two dog genes (L0C48433 1

AND L0C476394). In this clade, the absence of a chimpanzee or rodent counterparts to

these three genes suggests a loss in these species. for chimpanzee, however, loss by

pseudogenization is possibly involved (see physical maps described below); note that the

percentage of C2H2-ZNf genes annotated as pseudogenes was higher in chimpanzee that in

human C2H2-ZNf clusters (62, Supplernentary Table S4).

In group III, the relationship cf the four rodent genes with the dog and primate genes

could flot be resolved (bootstrap values < 95 ¾). However, a rodent-specific clade revealed

a mouse-specific duplication exhibiting a higher number cf C2H2-ZNF genes in mouse

than rat, as seen in several other cases in our study.

Superimposition of the phylogenetic trees with the physical maps of clusters

Comparison of gene trees, species tree and physical rnap infomiation cf cluster

19.12 genes and its syntenic homclogs provide better insights into the processes underlying

the evolution cf the C2H2-ZNF clusters. The phylogenetic tree obtained for cluster 19.12

(Figure 5) suggests a simultaneous differential expansion and loss of C2H2-ZNF genes

49

throughout evolution. In perfect agreement with the phylogenetic tree, genes of the

monophyletic groups I and III were found to be physically clustered together on the

chromosomes across mammals (Figure 6). Evidence for a tandem duplication event is

provided by the comparison of the relationship within C2H2-ZNF genes of Group I on the

tree with their spatial relationships in the physical maps that showed that the sequences of

the dog clade form a tandem array on the chromosome (Figure 5). In addition to tandem

duplication of individual genes within this group, e.g. cLOC484324 and cLOC484323

(figure 5) which are next to each other on the chromosome and exhibit the sarne orientation

(figure 6), we also discovered tandem duplication of multiple genes. for instance, three

genes (L0C482273, LOC6 11599, L0C480782/ orientation -, +, +) appear as a tandem

repeat ofthree other genes (LOC61 1583, L0C484328, L0C484326/ orientation -, +, +) in

this group (figures 5 and 6).

The group II mainly contains primate-specific C2H2-ZNF genes that cluster on the

phylogenetic tree in two well supported clades ( 97 % bootstrap) and a sub-group of

weaker support (93% bootstrap). Aimost ail these genes also cluster physicaily together on

the chromosome. Human orthoÏogy assignrnents for ten of the twelve chimpanzee genes

from group II (underlined in Figure 6) were corroborated by two lines of evidence i.e. from

the phylogeny, which was supported by the topology on the chromosome. furthermore,

genes from 7 out of 10 of the C2H2-ZNF ortholog pairs from this primate-specific cluster

exhibit the same number of zinc finger motifs and the same type of N-terminal motif

50

Species-specific variation in the number of finger motifs and the presence of N-

terminal conserved domains

When analysing the C2H2-ZNF genes from the 81 human clusters and their

syntenic homologs in mammals, we noticed that the average number of zinc finger motifs

varied depending on the C2H2-ZNF gene subfamiiies. Noticeably in ail the marnmaiian

species studied, genes with KRÀB and SCAN-KRAB motifs have a higher number of zinc

finger motifs tlian those from the other subfamiiies (Figure 7A). for exampÏe, member of

the KRAB subfamily have an average of 10 to 17 zinc finger motifs, whule members ofthe

BTB subfamily have oniy 2 to 3 (figure 7A). We also noted species-specific variation in

the number of zinc finger motifs within mammalian C2H2-ZNF genes. In particuiar, dog

tends to have a much higher number of zinc finger motifs in rnost C2H2-ZNF gene sub

families (Figure 7). Strikingly, L0C484264, a dog KRAB C2H2-ZNF gene exhibits 70 zinc

finger motifs which is to our knowledge the highest number of zinc finger motifs to be

reported for a zinc finger gene. Study of cluster 19.12 (Figure 5) iliustrates more

specificaliy the trend ofdog genes to exhibit more zinc finger motifs; the dog L0C484338

gene (group III), for example, lias six times more zinc finger motifs than its human

ortholog. Furtliermore, the dog gene L0C424326 lias neariy twice as many motifs as its

closest paralog L0C480782 (group I) (Figure 5). This indicates a quite recent and drastic

expansion of zinc finger motifs within dog C2H2-ZNF genes, after tlie separation of dog

from rodents and primates. In several cases, the C2H2-ZNf mammalian orthologs revealed

differences in tlieir numbers of finger motifs even within primate or within rodent lineages

(Figure 7B and Supplernentary Table 4).

51

In addition to the difference in the number of finger motifs in C2H2-ZNF

orthologs and paralogs, we also found a variation in the presence of the N-terminal effector

domains. As an example, orthologs ofthe C2H2-ZNF genes in the human cluster 6.2 show

a variation in the presence of the KRAB or SCAN domains (Figure 73), suggesting

frequent and multiple losses and/or gains of KRAB and SCAN domains during evolution.

To reconstntct these events, we analyzed in detail the exon-intron structure and sequences

ofthese genes (See Methods). Serendipitously, this analysis led us to the observation that a

large majority of the C2H2-ZNF containing a SCAN-KRAB or SCAN domain had each a

typical exon-intron organization (figure 7C). For example, both human genes, ZNF[92

(SCAN-KRAB) and ZNF187 (SCAN) and their respective orthologs in other species

(Figure 63) share the predominant exon-intron organization most typical of SCAN-KRA3

and SCAN C2H2-ZNF, respectively. Whule the dog LOC4$83 1$ bas only a SCAN dornain.

its coriesponding orthologs in human, mouse and rat have a SCAN-KRAB. When the

nucleotide sequence of the exon which would have been predicted to encode a KRAB

domain in dog (third exon after the SCAN) was compared with those ofhuman, mouse and

rat, the dog sequence exhibits a high conservation at the nucleotide level (>82 %) but no

significant similarity at the arnino acid ÏeveÏ. This indicates that the loss of the KRAB

domain in dog was due to sequence degeneration. Similarly, while the chimpanzee

ZNFÏ87 and its rat orthoÏog encode a SCAN dornain, a degenerate SCAN domain was

identified in the corresponding exon of their hurnan and mouse orthologs. For the human

SCAN-KRAB ZNF3O7 gene, we noticed that it exhibits an exon-intron organization typical

of SCAN-KRAB C2H2-ZNF (Figure 7C) whereas its orthologs in the chimpanzee, mouse

52

and rat encode solely a SCAN domain and present an exon-intron structure more typical

of SCAN C2H2-ZNf. However, it was found that, in chimpanzee, a sequence similar to the

KRAB sequence (99% at the nucleotide level) was embedded in the intron preceding the

exon encoding the zinc finger domain. No KRAB related sequence could be detected in the

rodent orthologs even with a detailed analysis of their sequences. Thus, either the KRAB

sequence was gain in the primate lineage or lost in the rodent lineage. For reasons

explained in the discussion, we believe that loss, rather than gain, is a more likely

hypothesis.

53

DISCUSSION

Comparative studies in genome research focused on the extensive similarities

existing between the human genome and the genomes from various other model organisms

which provide valuable insights into biological function and aetiology of human diseases.

However, differences existing among genomes have received less attention inspite of the

importance they may have in the physiological, morphological and behavioural distinctive

traits observed among species. A few studies on various gene families, such as the odorant

receptor family, pointed out to some differences existing between genes of closely rehited

species (Sitnikova and Su 1998; Lapidot, Pupe! et al. 2001; Niimura and Nei 2003; Gilad,

Man et al. 2005; Niirnura and Nei 2005). Our study ofthe C2H2-ZNf gene family reveals

that there is an extensive variation of the C2H2-ZNF gene content and organization in the

genornes from various mammals as well as in the domain composition of orthologous genes

arnong species. It also provides the first clear demonstration of the contribution of gene

loss in the C2H2-ZNF family during evolution which occurs at ifie level of clusters, genes

and their ftinctional dornains. We provide the first genome scale confirmation of the rapid

evolution of C2H2-ZNF gene clusters that occurs independently within related species

which also supports conclusions drawn from smaller-scale studies on individual genes,

clusters and C2H2-ZNF subfamilies (Dehal, Predki et al. 2001; Shannon, Harnilton et al.

2003; Harnilton, Huntley et al. 2006; Huntley, Baggott et al. 2006).

54

Substantial variation in the C2112-ZNF gene family size and clustering across

mammals

We report here the first complete catalogue of ail human C2H2-ZNF gene clusters and their

syntenic homologs in chimpanzee, mouse rat and dog. This catalogue reveals that in

hurnan, a large proportion of the genes from the C2H2-ZNF family (>70%) are organized

in clusters. Comparative studies of the five mammallan genomes indicated that the total

number of genes found in clusters varied considerably from 172 in rat to 518 in human

(number of genes found in clusters in human > chimpanzee > mouse > dog > rat).

Significantly, human and mouse have a larger number of clustered C2H2-ZNF genes

(>30%) as compared to chimpanzee and rat, respectively, indicating that independent

evolutionary events occurred after the divergence of the two primates (within the last 6-

10 million years) and two rodents (within 30-46 million years). We distinguish two kinds

of events: first, a variation in the niimber of C2H2-ZNF genes in syntenically hornologous

clusters and second, the existence of lineage- and species-specific clusters in primates,

rodents and canines. This can be accounted for by independent evolution of C2H2-ZNF

genes in these closely related species. Previous studies focusing on KRAB C2H2-ZNF

from chromosome 19 had identified and analyzed a primate-specific citister (Mohrenweiser

1998) including members of the primate-specific ZNF9I subfamily of C2H2-ZNf

(HamiÏton, Huntley et aÏ. 2006). Other studies on the KRAB C2H2-ZNf subfarnily

identified a differential expansion between a human KRAB C2H2-ZNF cluster and its

syntenic counterpart in mouse (Shannon, Hamilton et al. 2003) and more recently other

species-specific expansions based on draft of various mammalian genornes (Huntley,

55

Baggott et al. 2006). We illustrate and confïrrn at a larger scale the existence of an on

going process of genome dynamics with several lineage- and species-specific

rearrangements and continuous repertoire expansion taking place independently in ail

evolutionaiy branches, particularly in primates. This finding was only possible through the

analysis of a complete catalogue of ail the subfarnilies of C2H2-ZNf clusters and their

syntenic counterparts in mammals.

Gene duplication and loss: Two counteracting forces in the evolution of C2H2-ZNF

genes

An overview of $1 human C2H2-ZNF ciusters identified here revealed that a third

ofthern are pure clusters (with 2 to 24 C2H2-ZNF genes), i.e. they are flot interspersed with

other genes. Earlier observations of pure C2H2-ZNF gene ciusters have Ied to the

hypothesis that C2H2-ZNF genes in primates have expanded massively by tandem

duplication (Belleftoid, Marine et al. 1993; Eichler, Hoffrnan et al. 1998; Elemento and

Gascuel 2002; Schmidt and Durrett 2004; Bertrand and Gascuel 2005; Huntley, Baggott et

al. 2006). We revisited this question based on our catalogue of human C2H2-ZNF clusters

and their syntenic counterparts in chimpanzee, mouse, rat and dog. Here, we conflrmed

gene duplication and loss based on a reconciliation of both physicaÏ maps and the

surperimposition of gene trees onto the known species tree (Page and Charleston 1 997).

Our resuits clearly show that both gene gain and gene loss events have occurred multiple

times and independently in ail the mammals studied. Combined with physical map data, our

phylogenetic studies indicate that the expansion of C2H2-ZNF genes evidenced during the

56

evolution of the five species studied results from the combined action of single-gene

duplication and multiple gene duplication (for instances, duplication of ah or part of the

genes within a cluster). These duplication events were however counteracted by the loss of

individual genes or clusters as exemplified in several cases where related genes or clusters

found in primates and canine were absent in both or in any of the two rodents studied. This

study represents the first clear demonstration of the involvement of gene and cluster loss in

the evolution of C2H2-ZNF genes and suggests that during mammalian evolution the

duplication events outnumbered the loss events. Our resuits provide convincing support

that the C2H2-ZNF gene farnily evolved according to the “Birth and DeathT’ model as

proposed by Nei and colleagues (Nei, Gu et al. 1997; Nei 2000). According to this model,

new genes are created by duplication incÏuding tandem duplication and block gene

duplication (birth). While certain copies remain relatively unchanged in the genome for a

long tirne, others diverge functionahly by acquiring a new function. Sorne get deleted from

the genome or becorne pseudogenes following deleterious mutations (death through

ehimination or inactivation). In the case of C2H2-ZNF genes pseudogenization seems to be

limited, as suggested by expression studies and statistical analysis showing positive

seÏection based on the analysis of specific clusters (Schmidt and DmTett 2004; Huntley,

Baggott et al. 2006). This makes the C2H2-ZNf famlly different from the other gene

farnilies such as the olfactory receptor gene famiÏy (Glusman, Yanai et aÏ. 2001; Niimura

and Nei 2003) . Noticeably, gene loss by pseudogenization was prominent for the

olfactory receptors with humans accumulating a higher number of olfactoiy receptor

pseudogenes as compared to other primates and mouse (Sitnikova and Su 199$; Lapidot,

57

Pilpel et al. 2001; Niimura and Nei 2005). These variations in the numbers ofpseudogenes

and fiinctional genes have been associated with the differential chemosensoiy dependence

in these species (Sharon, Glusman et al. 1999; Quignon, Kirkness et al. 2003). In

comparison, besides the fact that they are known to fiinction as regulator of transcription,

the functions of only a few C2H2-ZNF proteins are known (Krebs, Larkins et al. 2003).

Further studies of C2H2-ZNf genes in mammals could shed light on the functional

consequences of different repertoires of these genes in different species. Until now, the

clustered organization of these genes lias made knock-out studies in animal modeis

inefficient, possibly due to redundancy. However, based on a better knowledge of the

organizationlcontent of C2H2-ZNF genes in the various genornes, large chromosornal

deletions of pure C2H2-ZNf clusters or other types of gene disruption or targeting

approaches could provide insights into the functions of these genes in different animal

models.

Evolution of C2H2-ZNF genes through gain and loss of finger motifs and N-terminal

effector domains

Evidence ofthe variation in the numbers of zinc finger motifs among orthologs was

previously reported for a subset of hurnan chromosome 19 C2H2-ZNf genes and their

mouse homologs (Looman, Abrink et al. 2002). It was shown that this variation is due to

both differential duplication of finger motifs and loss due to degeneration. In our study,

such variation in the number of zinc finger motifs arnong orthologs was observed

recurrently among ail mammals. Since the zinc finger motifs appears as a flexible motif

58

with the ability to bind DNA, RNA and/or proteins, changes in the zinc finger motif

sequences and number within C2H2-ZNF genes could differentially alter binding

specificities and thus protein function. Both changes in the number of C2H2-ZNF genes

and in the number of finger motifs encoded by orthologous genes may be determinant in

species-specific related ftmction.

The rapid evolution of the C2H2-ZNF genes observed in the mammalian lineage

was flot limited to the variation in the number of genes and zinc finger motifs. Variation in

the presence of N-terminal effector domains, such as SCAN or KRAB, was observed in

orthologs and could be accounted for by either gain or loss of these motifs in the various

species. Loss by sequence degeneration of both the SCAN and the KRAB sequences vas

confirmed in several cases in our study. In some cases, neither loss nor gain could be

resolved. A puzzling question remains however if one assumes that gain of KRAB and

SCAN sequences can occur recurrently within C2H2-ZNF genes. It is indeed diffictiit to

explain that these effector domains are always found in association with and N-terminal to

the zinc finger motifs of C2H2-ZNF proteins and that the SCAN dornain is aiways

positioned N-terminal to the KRAB domain of SCAN-KRAB C2H2-ZNF proteins.

Interestingly, by analyzing the exon-intron structure of C2H2-ZNF genes from the clusters,

we found that most SCAN C2H2-ZNf and SCAN-KRAB C2H2-ZNF genes have each a

typical exon-intron structure (Figure 7C and Figure 8A). This suggests that the acquisition

of a SCAN and KRAB sequences by C2H2-ZNF genes corresponds most likely to singular

events. This led us to propose the moUd described in Figure 8. Considering that the SCAN

domain is found in ah vertebrates and is more ancient than the KRAB dornain only found

59

in tetrapods, we suggest that first a SCAN-C2H2 ZNF gene was formed in an ancestor of

vertebrates through the gain of SCAN sequence and that later, after the emergence of the

tetrapods, the gain of a KRAB sequence by a SCAN C2H2-ZNF gene gave rise to a SCAN

KRAB C2H2-ZNF gene (Figure 83). These two gain events possibly occurred through an

exon-shuffling mechanism. Diversification of the C2H2-ZNF repertoires from each

subfamily then occuned dynamicaliy through on-going gene duplications and loss by

deletion or degeneration of the SCAN and!or KRAB sequences. As irnplied by this model,

the birth of the SCAN-KRAB C2H2-ZNF subfamily occurred earlier than that of the

KRAB C2H2-ZNF subfamiiy. This was consistent with previous data showing that SCAN

KRAB-ZNf genes do flot group together on one evolutionary ciade but intermix with

KRAB-ZNF genes in phylogenetic trees of the KRAB sequence (Looman, Abrink et al.

2002; Huntley, Baggott et al. 2006) (Huntiey, Baggott et al. 2006). On the whole, our

model is in agreement with the fact that C2H2-ZNF orthologs often belong to different

C2H2-ZNF subfamilies and that we observed intermingling of C2H2-ZNf genes from the

SCAN, KRAB and SCAN-KRAB subfamiiies in many C2H2-ZNF clusters. Our study

clearly indicated that the evolution of C2H2-ZNf subfamilies is tightly iinked and stresses

that the assignrnent of proper orthology requires comprehensive analysis of ail C2H2-ZNF

genes rather than the individual analysis of specific C2H2-ZNF subfamilies. It also points

to the importance of ioss/contraction and secondary simplification whose role in the

dynamics of evolution is ofien underestirnated. The underiying rnechanisms in the

expansion of C2H2-ZNF genes and the flinctional consequences of the important changes

(gain and loss) occurring in their repertoires of various mammals are unclear. These

60

variations, for example, may be at the advantage of complex organisrns by providing more

subtie and species-specific control in gene expression for morphogenesis or cognitive

functions.

ACKNOWLEDGEMENTS

The authors thank Franz B. Lang for suggestions and help in phylogenetic analysis and

Nicolas Lartillot (Universite de Montreal) for constructive comments on tree building and

datasets. We also acknowledge Herve Philippe, Henner Brinkmann and Amy Hauth for

critical discussions and Allan $un for assistance in hardware and software installations.

This work was supported by a grant from Natural Sciences and Engineering Research

Council of Canada (to M.A). M.A is a Chercheur National from the Fonds de la recherché

Sante du Quebec (FRSQ) and G.B is an associate from CIFAR (Canadian Institute for

Advanced Research).

61

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68

Figure 1: Flowchart of the analysis procedure ofC2H2-ZNF genes and clusters

Compare with syntcnicaliv homologous

Identify C2H2-ZNF elusters elusters from complcted genomes

I IAna; in

ht:pe1ulus

tttisrvegicus inisiliiris

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______________________

thc homologous C2H2-ZNF clusters

Extract compicte dataset of C2112-ZNF gcncs

IRcCOflcilllltiofl of phylogenetic rehitionships

+ spatial relationships + Species Trec

ipiens

_______________

69

350

300

° 250u

200

150Q

100

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No. of C2H2-ZNF genes

Figure 2: Distribution of ail the singletons and clustered genes from the various

human C2112-ZNF sub-families ami gene composition of the C2112-ZNF clusters

A

45% 43 %O Singleton

• In clusters

5.7 %

, _ , s,Type 0f domains associated with C2H2-ZNF genes

B

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40

35

20

15

10

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C2H2-ZNF clusters with:

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70

figure 2

Distribution of ail the singletons and clustered genes from the varlous human C2112-

ZNf sub-families and gene composition of the C2H2-ZNf clusters.

A) The number of genes belonging to the various C2H2-ZNF subfamilies are shown as

weÏÏ as the proportion of genes found as singletons or as part of clusters. C2H2-ZNF genes

associated with KRAB and SCAN domains are more often found to be ctustered. S-K=

C2H2-ZNf containing both a SCAN and a KRAB domain. NONE= C2H2-ZNf without

any conserved domain associated. The percentage distribution is mentioned on top of each

bar for each sub-family.

B) The number of C2H2-ZNf clusters is shown with respect to the number of genes present

in each cluster. The proportion of clusters composed of solely C2H2-ZNf without any

intervening gene or with intervening genes other than C2H2 ZNf (Non-C2H2-ZNF) is aÏso

represented. A star (*) identifies large clusters present on chromosome 19.

71

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Figure 3

Differential expansion and Ioss of C2H2-ZNF clusters in five mammalian genomes.

A) Evolution of the C2H2-ZNF repertoires in primates, rodents and dog. The number of

C2H2-ZNF clusters and the total number of C2H2-ZNF found in these clusters are

mentioned on the species tree. Since Xenopus taevis and Galtus gaÏÏus C2H2-ZNF are used

as an outgroup in phylogenetic studies, these species are also positioned on the tree.

The figure indicates the primate-specific increase in the number of C2H2-ZNf as cornpared

to rodents and dog.

B) A graphical representation of different scenarios seen in the evolution of human C2H2-

ZNF clusters and its syntenically hornologous C2H2-ZNf clusters in chirnpanzee, mouse,

rat and dog. The human clusters selected and narned on the graph as well as their syntenic

counterparts were 1) present in ail species, 2) primate-specific, 3) lost in rodents or 4)

absent in dog. For each hurnan C2H2-ZNF cluster named on the graph, the first number

indicates the chromosome number and the second is the number attributed to that cluster on

the chromosome. Supplementary Figure S2 provides a more comprehensive graphical

representation including the 40 human clusters that contain at least 3 C2H2-ZNF and their

syntenic counterparts in the four other mammals.

73

Figure 4: Evolutionary scenarios in the phylogenetic tree

A C

Species Tree Cene Ioss in species

Species I SI gene

Species 2 S2 ene

Species3 •••• S3eiie

Species 3 54 gene

outgroup Outgroup

BCene gain in species

74

figure 4

Evolutionary scenarios in the phylogenetic tree

The different kinds ofevolutionary scenarios seen in the phylogenetic tree are shown.

A) Species tree showing the evolutionary relationship between the species, 1, 2, 3 and 4.

B) A species-specific gain of genes appears as a clade including a single hornolog from

one species and multiple homologs from the other. Phylogeny between genes Si gene, S2

gene, 53 gene and S4 gene from species 1, 2, 3 and 4 respectively. Gene gain in species 4 is

observed. C) Species-specific gene loss appears as the absence of a corresponding ortholog

for one species on the tree and is deduced from the evolutionary relationships of the species

considered with the other species. Loss ofthe corresponding gene (S3 gene) in species 3.

75

Figure 5: Phylogenetic analysis of C2H2-ZNF genes in cluster 19.12 of human and its

syntenic counterparts in other mammals

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Figure 5

Phylogenetic analysis ofC2H2-ZNF genes in cluster 19.12 ofhuman and its syntenic

counterparts in other mammals.

A phylogenetic tree was built using the amino acid sequences corresponding to the zinc

finger regions of the various human C2H2-ZNF from cluster 19.12 and their syntenic

counterparts in chirnpanzee, mouse, rat and dog. The tree was generated using a maximum

Ïikelihood method (RaxML) and verified using a bayesian method (Mr.Bayes). 346 sites

from 101 sequences (including the 20 outgroup sequences from chicken and Xenopus) were

used in the analysis. The tree is divided into three major Groups (I-III). A tabulation of the

number of genes present in each group is indicated for cadi species (h: human, p:

chimpanzee, rn: mouse, r: rat, e: dog. lie bootstraps values are indicated for each node on

the tree. A small black circle is also represented at each node in cases where the posterior

probability value is equal to 1.00. Tus cluster contains only C2H2-ZNF genes that are

either from tic KRAB subfamily or that do not encode any conserved N-terminal domain.

Next to the name of each C2H2-ZNF gene, the presence of an N-terminal KRAB dornain is

indicated by a K and number of zinc finger motifs is mentioned. A clear evidence of

differential expansion is seen in primates and dog. Loss of C2H2-ZNF in the rodent lineage

is also observed.

77

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Figure 6

Physical maps showing the organization ofthe human C2112-ZNF from cluster 19.12

localized on 19q13.4 and its syntenïcally homologous counterparts in other mammals.

For the large C2H2-ZNF cluster 19.12 and its syntenically homologous counterparts in

chirnpanzee, mouse, rat and dog, each C2H2-ZNf genes is represented by an open arrow

which indicates its orientation on the chromosome strands; this exciudes the pseudogenes

whose name appears in parenthesis. For these clusters which contain only C2H2-ZNF that

are from the KRAB subfamily or that do flot encode any conserved N-terminal domain, the

presence of a conserved N-terminal KRAB dornain is indicated by as square positioned in

front ofthe open arrow representing the gene. Genes identified as orthologs, based on the

phylogenetic tree and physical rnaps, are underlined and are aligned verticaÏly on their

respective chromosomes. Dotted lines separate the genes belonging to Group I, Group II

and Group III defined in the phylogenetic tree (figure 5). The two species specific groups

from dog and primates are seen in Group I and Group II, respectively.

79

Figure 7: Variation in the numbers of zinc finger motifs in mammals and in the

presence of conserved N-terminal domains in orthologs

•Human Chimpanze Mouse JRat uDog

r -

ii 1H F ii a [ r

Ail lRAll 5C ‘. - (RAIl %( lii li I lIt)lE I) l)

C2H2-ZNF subfamilies

B

(--

Humati

___________________

2 —

E E

Chimpanzee

Mouse

---------------N R N N N

N N N N N

Rat

Dog

CHuman C2H2-ZNF from clusters

SCA-KRAll(II/14)

SCAN(16/29)

80

figure 7

Variation in the numbers of zinc finger motifs in mammals ami in the presence of

conserved N-terminal domains in orthologs.

A) The average number of zinc finger motifs was calcuiated for ail the C2H2-ZNF from the

$1 hurnan clusters identified and their corresponding syntenically liomologous clusters in

the other mammals; for each species, the average number for the total C2H2-ZNF (Ail) and

for members ofthe various C2H2-ZNF sub-families (KRAB, SCAN, SCAN-KRA3, 3TB,

HOMEO, SET and, NONE no conserved domain associated) is presented. For each

categoiy, the number of genes in each species is listed above the bars in the following order

(human, chimpanzee, mouse, rat and dog).

B) for the human C2H2-ZNF cluster 6.2 (chromosome 6p22. 1) and its syntenically

homologous counterparts in chirnpanzee, mouse, rat and dog, each C2H2-ZNF genes is

represented by an open arrow whicli indicates its orientation on the chromosome strands;

this exciudes the pseudogenes whose name appears in parenthesis. For these clusters which

contain C2H2-ZNF that are from the KRAB or SCAN subfamily or that do not encode any

conserved N-terminal domain, the presence ofa conserved N-terminal is indicatcd by as

square for a KRAB domain or an open circle for a SCAN dornain both being positioned in

front of the open arrow representing the gene. Genes identified as orthoÏogs, based on the

phylogenetic tree and physical maps, are aiigned vertically on their respective

chromosomes. Cases wliere domain shuffling was observed among orthoÏogs from the

different mammals are marked by a grey box.

$1

C) Exon-Intron organization of most hurnan C2H2-ZNF from the SCAN-KRAB and

SCAN subfarnilies. 80% ofthe human SCAN-KRAB C2H2-ZNf (11/14) and 55% ofthe

SCAN C2H2-ZNF (16/29) found in clusters have the presented exon-intron structures

shown. The exons encoding the SCAN, KRAB (A box) and ZNF are indicated.

82

Figure 8: Model for the evolution of the SCAN, SCAN-KRAB and KRAB C2H2-ZNF

subfamilies

AC2112-ZNf SCAN-C2H2-ZNf SCAN-KRAB C2H2-ZNF KRÀB C2H2-ZNF

Gain afSCAN event

Gtii,, ofKRAB avent

E1Loss cfSCAN

B

F RAB ZNF NF

Vcrtehrate-spccftîc

Tetrapod-specific

Dup Dup Dup

2Singular gain events

Gain of SCAN Gain ofKRABZNF SCAN-ZNF SCAN-KRABZNF 4

Ï L55OfKB] OMultiple loss cventst Loss of SCAN

KRAB-ZNF

Lois of SCAN-KRAB

______

Liii ofKRAB

83

Figure 8

Model for the evolution of the SCAN, SCAN-KRAB and KRAB C2112-ZNF

subfamilies

A) Sequential events of exon shuffling leading to the birth of SCAN-C2H2-ZNF and

SCAN-KRAB C2H2-ZN}’ subfamilies. Most of the SCAN-C2H2-ZNF and the SCAN

KRAB C2H2 ZNF have the exon-intron structure shown (boxes represent exons). Birth of

new families may have occurred by an exon shuffling mechanism leading presumably first

to the acquisition of a SCAN domain by a C2H2-ZNF and later of a KRAB domain by a

SCAN-C2H2-ZNF. Most SCAN-KRAB C2H2-ZNF have a single exon placed in between

the exon encoding the KRAB A box (identified as KRAB) and the exon encoding the zinc

finger domain (ZNF). This exon encodes in most instances the so-called KRAB B, b, or C

boxes.

B) Dynamic evolution of C2H2-ZNF after birth of the SCAN and $CAN-KRAB

subfamilies through gene duplication and recurrent loss of effector domains. A first SCAN

C2H2-ZNF appeared in an ancestor of vertebrates following the gain of a SCAN domain by

a C2H2-ZNf (in grey box); duplication then led to the establishment of the SCAN C2H2-

ZNF subfamily. The gain of a KRAB domain at the emergence of tetrapods by a SCAN

C2H2-ZNF gave rise to a SCAN-KRAB C2H2-ZNf (in grey box). This was followed by

duplication and establishment of the SCAN-KRAB subfamily. Loss of SCAN domain by

deletion or degeneration from some SCAN-KRAB C2H2-ZNF genes followed in many

instances by duplication led to the expansion of the KRAB C2H2-ZNF. Duplication and

loss of SCAN or KRAB domains by deletion or degeneration from SCAN, $CAN-KRÀB

84

and KRAB C2H2-ZNF subfamilies are seen as a recurrent theme shaping the repertoires

ofthe C2H2-ZNF subfamilies.

85

98Totalno.ofhumanC2H2-ZNFgenes

,.oor)oooOOOO

rrI

10

40

80-t

200

400

-t

600

800

1000—.

-t1400

1800(s—.

oCI)

3000

w

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7000

9000—J

oc11000

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15000

17000

20000

22000

27000C

35000

Supplementary Figure 1: Distribution of intergenic distances between 718 C2112-

ZNF in the human genome.

Supplementary Figure 1

Distribution of intergenic distances between 71$ C2H2-ZNF in the human genome.

The intergenic distances between the consecutive C2H2-ZNF on each chromosome was

calculated for each C2H2-ZNF ofthe human genome. For the 718 C2H2-ZNF, the number

of C2H2-ZNF found within the range of intergenic distances indicated on the x axis is

plotted on the y axis. For example, there are 108 C2H2-ZNF within 10 to 20 Kb from a

consecutive C2H2-ZNF.

$7

Supplementary Figure 2: Comparison of the number ofC2H2-ZNF genes in the 40

human clusters containing at Ieast 3 C2H2-ZNF ami their syntenic counterparts in

four other mammals

Supplementary Figure 2A

9-

8

Human 1Chimpanzee DMouse Rat DDog,w 7 -

__________

ew

6e

£

w 5ie£w

4Nc’1

ii IL k b1,2 2,2 3,1 3,2 4,1 7,2 7,3 7,6 8,3 9,1 10,2 10,3 15,2 15,3 17,1 18,1 19,2 19,10 20,2 X.1

Syntenically homologous C2H2-ZNF clusters with 3-5 C2H2-ZNF in Human

88

Supplementary Figure 2B

80

70

Human S Chimpanzee D Mouse Rat D Dog60

(n

50

40

uzN

30(.4oqo 20

:Lh,hk_idb_Lii, L1,5 3,3 5,1 6,2 7,4 7,5 7,7 8,4 10,1 12,1 16,1 16,3 19,5 19,6 19.7 19,8 19,9 19,11 19.12 19,13

Syntenically homologous C2H2-ZNF clusters with at Ieast 6 C2H2-ZNF in Human

89

Supplementary Figure 2

Comparison of the number of C2H2-ZNF genes in the 40 human clusters containing

at least 3 C2H2-ZNF and their syntenic counterparts in four other mammals.

For each human C2H2-ZNF cluster named of the graph, the first number indicates the

chromosome number and the second is the number attributed to that cluster on the

chromosome. C2H2-ZNF chisters with six or more (A) and three to five (B) genes in

human and their syntenic counterparts Chimpanzee, Mouse, Rat and Dog. This figure

provides evidence of C2H2-ZNF differential species-specific expansion and gene loss in

rodents.

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Tad

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Supplementary Table S2

Comprehensive summary of the organization of ail C2H2-ZNF tound as singletons or in clusters

on each human chromosome and classified with respect to the various C2H2-ZNF sub-families.

C2H2-ZNF sub-families

Total No. C2H2-ZNF in KRAB SCAN-KRAB SCAN BTB SET HOMEC oChr No. C2H2-ZNP No. Clusters Ciusters S C S C S C S C S C S C S C

1 36 6 17 1 9 1 1 7 1 9 7

2 17 3 7 1 2 9 5

3 30 6 20 2 5 3 2 1 6 11

4 10 1 4 1 2 1 4 2

5 15 1 6 1 5 8 1

6 28 3 16 1 2 2 732 8 3

7 47 7 41 127 2 512

8 30 4 16 36 1 1 2 10 8

9 23 4 10 5 3 1 3 2 4 5

10 22 3 13 8 9 5

11 15 2 4 3 1 1 1 3 4 2

12 15 1 7 2 6 : 6 1

13 5 2 4 1 4

14 10 2 4 1 2 :i 5 1

15 12 3 8 12 1 3 5

16 33 6 27 10 1 2 3 5 12

17 13 2 5 1 1 1 1 11 5 2

18 14 2 6 1 32 5 3

19 289 13 279 3 185 1 13 4 1 6 76

20 18 3 7 2 2 1 2 6 5

21 3 1 2 1 1 1

22 10 1 2 2 2 4 2

X 19 4 11 1 6 1 11 6 3

Y 4 1 2 : 2 2

Total 718 81 578 32 280 4 14 3 30 28 13 1 1 2 3 130 177

No. of C2H2-ZNF without an encoded conserved N-terminal domain (ø), found as singletons (S)

or in C2H2-ZNF clusters (C)

Tadepally et.aI 172


Gene organization of the 81 human C2H2-ZNF clusters.

Cluster1

No. 0f Clusters with Order of the genes from the

Position C2H2-ZNF Solely C2H2-ZNF different C2H2-ZNF subfamilies2 Cluster composition4

1.1

1.2

1.3

1.4

1.5

1.6

2.1

2.2

2.3

3.1

3.2

3.3

3.4

3.5

3.6

4.1

5.1

6.1

6.2

6.3

7.1

7.2

7.3

7.4

7.5

7.6

7.7

8.1

8.2

8.3

8.4

9.1

9.2

9.3

9.4

10.1

10.2

10.3

11,1

11.2

12.1

13.1

13.2

14.1 j14.2

15.1

15.2

15.3

16.1

1p36.l1

1p34.2

1p34.1

1q42.13

1 q44

1 q44

2q 11.1

2q1 3

3p22.1

3p2l .32

3q1 3.2

3q24

3q26.32

4pl 6.3

5q35.3

6p22.1

6p22.1

6p2l.3

7p22.1

7p1 1.2

7q1 1.21

7q22.1

7q22.1

7q36.1

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8q24.1 3

8q24.3

8q24.3

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9q31 .2

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9q33,2

lOpll.21

lOqi 1.21

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llpl5.4

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12q24.33

13q22.1

13q32.3

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1 4q23.3

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1 5q26.1

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2

3

2

2

6

2

2

3

2

3

3

8

2

2

2

4

6

2

12

2

2

4

5

9

8

3

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2

5

7

4

2

2

2

6

3

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7

2

2

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2

2

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9

No

No

No

No

No

Yes

No

Yes

No

Yes

No

Yes

No

YesNo

Yes

No

No

No

No

No

No

No

No

No

YesNo

No

No

No

No

No

No

Yes

Yes

Yes

No

Yes

Yes

Yes

Yes

No

YesYes

YesNo

No

No

No

0/0

KIKIK

0/0

o/K

KJKJK/KIK/S-K

0/0

0/0

K’K/ø

0/0

o/KIK

0/1<1K

S-KIKIS-KJø/S-KIo/ø/ø

ø/B

0/0

0/0

KIø/ø/K

K/K/ø/KJK/K

ø/K

S/S/S-K/ø/S/S-K/o/S/S/S/S/K

B/B

o/K

ø/K/ø/K

o/o/KIø/K

KJK/K/ø/ø/K/K/ø/K

K/S-K/S-KIø/ø/KIK/K

K/K/K

/K/K/K/K/ø/K-K

0/0

H/H

ø/ø/o/ø/K

ø/KIKJKIKIo/K

ø/KJKJø

0/0

o/K

BIB

KIø/ø/KJK/K

o/KIK

K/ø/K/ø

S-K/K

0/0

KJKIKJKJKJKIø

0/0

0/0

H/o

BIB

B/o

S/S/ø

0/0/0

S/KIS-K/ø/S-KIKIø/S/o

Pure

Pure

Pure

Mixed

Mixed

Pure

Pure

Mixed

Pure

Mixed

Mixed

Mixed

Mixed

Pure

Pure

Mixed

Mixed

Mixed

Mxed

Pure

Mixed

Mxed

Mixed

Mixed

Mixed

Pure

Mixed

Pure

Pure

Mixed

Mixed

Mixed

Pure

Mixed

Pure

Mixed

Mixed

Mixed

Mixed

Pure

Mixed

Pure

Pure

Mixed

Pure

Mixed

Mixed

Pure

Mixed

Tadepally et.aI 113

16.2 16pI3.3 2 No øIS Mixed

16.3 16p11.2 10 No øIø/5K/ø/ø/ø Mixed

16.4 l6pll.2 2 No øIK Mixed

16.5 16q22 2 Yes KIK Pure

166 16q24 2 2 Yes oie Pure

17.1 17p13.2 3 No ø/S/ø Mixed

17.2 l7pll.2 2 No S-K/K Mixed

18.1 18q12 4 Yes S/o/S/S Mixed

18.2 18q23 2 No øiø Pure

19.1 19q13.2 3 Yes K/KJK Pure

19.1 l9p13.3 2 No ø/B Mixed

19.11 19q13.31 24 Yes 19KI5ø Mxed

19.12 19q13.41 43 No ‘ 30KI13ø Mixed

19.13 19q13.43 76 No 343K/1S-K/12S/1B/19ø Mixed

19.2 19p13.3 5 Yes K/K/K/K/K Pute

19,3 19p13.3 2 No ø/B Mixed

19.4 19p13.2 2 No Kb Mixed

19.5 lgpl3.2 14 No 9K/5ø Mixed

19.6 19pl3.2 28 No 21K/7ø Mixed

19.7 19pl3.ll 40 No 328K/12ø Mixed

19.8 19q13.11 8 No 3K/S/3ø Mixed

19.9 19q13.12 32 No 23K/1B/8ø Mixed

20.1 20p11.23 2 No K/ø Mixed

20.2 20q13.12 3 No obøbK Mixed

20.3 20q13.2 2 Yes øÏø Pure

21.1 21q22.3 2 No Se/B Mixed

22.1 22q11.22 2 No 0/0 Pure

X.1 Xpl 1.23 5 No K/K/K/K/K Pure

X.2 Xpll.1 2 No 0/0 Pure

X.3 Xq26.3 2 Yes S-K/S Mixed

X.4 Xq28 2 No oiK Mixed

Y.1 Yql 1.23 2 No 0/0 Pure

Total 81 518 Yes= 25; No = 56 Pure= 29; Mixed= 52

Clusters C2H2-ZNF

For the cluster name, the first number correspond to the chromosome on which the cluster is found

and the second to the number attributed to the cluster.

2 Sequential order 0f the genes from the different C2H2-ZNF subfamilles such as KRAB (K), SCAN (S),

SCAN-KRAB (S-K), SET (Se), HOMEO (H) and without an encoded conserved N-terminal domain (o)

For the very large clusters, the number of C2H2-ZNF from each subfamilles is specified

(eg: 23 K means that 23 consecutive genes from the KRAB-C2H2-ZNF subfamily are found in the cluster).

‘ Pure’ = The cluster is composed 0f C2H2-ZNF from a single subfamily;

‘Mixed’ = different subfamilles 0f C2H2-ZNF are present in the cluster.

Note: ‘Pure’ clusters with solely tandemly repeated C2H2-ZNF are in grey

Tadepally et.aI 114

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Tad

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Compreliensive catalogue of the C2H2-ZNF genes from the 81 human clusters and

their syntenically homologous clusters from other mammalian genomes (Chimpanzee,

Mouse, Rat and Dog).

For the 81 human (h) C2H2-ZNF clusters and their corresponding syntenically homologous

clusters in chirnpanzee (p), mouse (ni), rat (r) and dog (c), this table provides the cluster

number, the position on the chromosome, the flanking genes (FG), the names ofthe C2H2-

ZNF from the cluster (in bold), the domain associated (D) (K KRAB, S = SCAN, S-K=

SCAN-KRAB, B = BTB, H = HOMEO, Se = SET and ,‘cJ ‘ no domain associated), the

number of zinc finger motifs present (F), the orientation (O).

144

Chapter 3. DISCUSSION

145

Many studies in biology focus on the extensive similarities between the genomes of

human and model organisms, to extract insights into the molecular mechanisms and

aetiology ofhuman diseases. Our investigation ofthe C2H2-ZNF gene family in mammals

reveals that there is an extensive variation ofthe C2H2-ZNF gene content and genomic

organization as well as the domain composition of orthologous genes among species. In

addition, our study is the first to provide a clear demonstration of the important contribution

of gene loss in the evolution of C2H2-ZNf family and to demonstrate the rapid evolution

of C2H2-ZNf genes that occurs between related species, our observations at the genomic

scale provide insights into C2H2-ZNF gene evolution that confirm conclusions drawn from

smaller-scale studies on individual genes, clusters and C2H2-ZNF subfamilies.

The major contributions of our study are:

j. The extensive anaiysis of ail the C2H2-ZNF genes in the human genome.

ii. A comprehensive and systematic anaiysis of ail the human C2H2-ZNF clusters

and the identification of their syntenicaÏiy homoiogous counterparts in other

mammalian genomes.

iii. The distinction of species-specific expansion and Ioss in C2H2-ZNf clusters

and genes in ail mammals.

iv. The identification of variation in the number of zinc finger motifs and the

presence or absence of the conserved N-terminal domains associated with

C2H2-ZNF mammalian orthologs.

146

y. The tracing back of different evoiutionary patterns of the C2H2-ZNF gene

family within primates and rodents.

vi. The establishment of a mode! reconstructing the history and evolution of the

SCAN, SCAN-KRAB and KRÀB subfamilies.

In brief, our study reveals that the multiple and independent duplications and !osses of

C2H2-ZNf genes and their effector domains within different lineages and species has

shaped and diversified C2H2-ZNf repertoires in mammals.

3.1 The C2H2-ZNF genes in the human genome

Earlier studies of C2H2-ZNF genes focussed on human chromosome 19 (Eichler,

Hoffman et al. 199$; Dehal, Predki et al. 2001; Looman, Abrink et al. 2002; Shannon,

Hamilton et al. 2003) and KRAB C2H2-ZNf subfamily in human (Huntley, Baggott et al.

2006). In contrast, our study provides a comprehensive and systematic analysis of ail the

C2H2-ZNF genes (Supplementary Table S Ï) in the human genome. We identified and

analyzed the organization of 718 C2H2-ZNf genes in the human genome and classified

them into different subfamilies of C2H2-ZNF (KRAB-C2H2-ZNF, SCAN-C2H2-ZNF,

BTB-C2H2-ZNF and those without a conserved N-terminal domain). We also discovered

two new C2H2-ZNF subfamilies, the HOMEO and SET subfamily which have a limited

number ofmembers (5 and 2, respectively) possibly due to a more recent appearance or to a

different rate of duplication and loss.

147

Consistent with previous reports (Rousseau-Merck, Koczan et al. 2002; Huntley,

Baggott et al. 2006), we observed a massive clustering of the C2H2-ZNF on the human

genome. More than 70% of the genes are organized into clusters on the human genome.

However, in addition to the earlier reported clusters on human chromosome 19, (Venter,

Adams et al. 2001; Huntley, Baggott et ai. 2006), we also located a substantial amount of

ciusters (83%) on the other chromosomes ofthe human genome (Supplementary Table S2).

Interestingly, the distribution of C2H2-ZNF genes is positively biased toward chromosome

19, harbouring 40% of ail C2H2-ZNf genes in humans. Most of the human C2H2-ZNF

genes are organized into clusters (500) with more than 60% of these clusters containing

intermixed sets of genes from different subfamilies (Supplementary table S3).

The above observations were only possible through the study of all the C2H2-ZNF

sub-families at the whole genome level.

3.2 Variation in the numbers of C2112-ZNF genes in

mammalian clusters

A systematic and comprehensive analysis of the human C2H2-ZNF clusters and its

syntenic counterparts in the chimpanzee, mouse, rat and dog genomes, allowed us to gain

insights into the evolution of ah the C2H2-ZNF gene subfamilies in mammals

($uppiementaiy Table S4). The criterion to identify homologous clusters in syntenic

regions was based on the flanking genes identified for each human cluster. Interestingly,

this analysis revealed a high variation in the number of C2H2-ZNF genes within

14$

syntenically homologous clusters of mammals. Considering primates, humans have 518

C2H2-ZNF forming $1 clusters whereas chimpanzee has only 397 C2H2-ZNF organized

into 79 clusters. This suggests that almost ail the C2H2-ZNf clusters in human have a

syntenic counterpart in chimpanzee. However, humans have 30 % more C2H2-ZNf genes

within the identified clusters than chimpanzee implying that C2H2-ZNF genes are evolving

differently within the primate lineage. A similar pattem was observed within the rodent

lineage, where mouse and rat have 232 and 172 C2H2-ZNf organized into 62 and 58

clusters, respectively. A differential expansion of C2H2-ZNF genes, particularly striking in

primates was evident in mammals (human>chimpanzee>mouse>dog>rat for the number of

genes within clusters and human>chimpanzee>mouse>rat>dog for the number of clusters)

(Figure 2 and Supplementary Table S4). A doser look at the individual syntenic clusters in

mouse, rat and dog indicates many cases where dog has more number of genes than rodents

and more specifically than rat (Supplementary Figure S2).

Our study indicates that C2H2-ZNF genes are indeed rapidly evolving genes as

evident for example within the primate and rodents lineages. The differential expansion

observed in the various species may be accounted both by differential duplication and/or

loss. If the high numbers of C2H2-ZNF genes found in human as compared to chimpanzee

suggest an expansion specific to human through tandem duplication, it seems that this

difference is not solely due to duplications but also involves loss in chimpanzee as seen in

many gene families (Fortna, Kim et aI. 2004). In agreement with this, there are more

pseudogenes in chimpanzee clusters (as annotated in Genbank) than in the corresponding

human clusters. Thus, the variation in the numbers of C2H2-ZNF genes observed within

149

primates could be attributed to both gene duplication and loss due to deletion or

pseudogenization, gain being more predominant in human and loss in chimpanzee.

Interestingly, a variation in the number of C2H2-ZNF genes is also evident in rodents.

However, in this case in almost ail the clusters, mouse has either a higher or equal number

of C2H2-ZNF as compared to rat. Altogether, our study suggests that in addition to lineage

or species-specific increase in the numbers ofthe C2H2-ZNf genes, loss ofthese genes has

also played a very important role in the evolution of this gene family. The evaluation of the

relative contribution of gene duplication and loss requires detailed phylogenetic studies.

3.3 Evolution of C2112-ZNF genes in mammals through

differential expansion and loss

Phylogenetic analyses of human C2H2-ZNF clusters with their syntenic

counterparts from other mammals provided a better estimation of the relative contribution

of gene duplication and loss in the analyzed clusters.

For example, the phylogenetic analysis of the C2H2-ZNF genes from human cluster

Ï 9.12 and its syntenically homologous clusters in mammals combined with the physical

maps of these clusters gives us insights into the gene rearrangement mechanisms that could

have taken place during evolution. Consistent with previous individual reports of lineage

specific expansion, more specifically of KRAB C2H2-ZNF genes (Dehal, Predki et al.

2001; Shannon, Hamilton et al. 2003; Huntley, Baggott et al. 2006), a primate lineage

specific expansion but also a dog specific expansion and a mouse duplication of C2H2-

150

ZNF genes were clearly identified . In ail species, tandem dupiication was found

responsibie for the species-specific increase in the number of C2H2-ZNF genes as

confirmed by the fact that the genes that group together in the tree are almost aiways

physicaiïy clustered together in the cluster on the chromosome. Furthermore, the

orientations of the genes belonging to the same ciade in the phylogenetic tree and their

orthoiogs were almost aiways the same. In a few instances, however, the orientations of the

genes belonging to the same phyiogenetic clade were different and genes within syntenic

clusters were inverted in a few instances, revealing a lot ofpossibie gene rearrangements.

Clear evidence of gene loss was aiso obtained by analyzing cluster 19.12 and other clusters.

Considering that rodents are evolutionariiy more related to primates than dog, an absence in

rodents of C2H2-ZNF ciusters or genes, that are present in primates and dog, suggests a

loss in rodents. Severai examples of genes loss in rodents were obtained. The phylogenetic

analysis also indicated loss of genes in chimpanzee by pseudogenization as suggested

above.

Aitogether our studies indicate a predominant role of gene gain by tandem duplication over

gene loss for the evolution of C2H2-ZNF genes in mammals. It should however be pointed

out that more definitive conclusions about the pseudogene status of the various C2H2-ZNF

genes and on the role of these genes requires detaiied functional investigation of the

individual genes.

151

3.4 Evolutïon of the C2H2-ZNF genes tlirougli duplication or

loss of zinc linger and N-terminal effector motifs

In accordance with a previous study on the average number of zinc finger motifs

from a few plant (1), yeast(1.5), nematode (2.5), insect (3.5) and human (8) C2H2-ZNF

(Venter, Adams et al. 2001), an in depth analysis of the zinc finger motifs associated with

the C2H2-ZNf genes found in clusters in human and their syntenic genes in chimpanzee,

mouse, rat and dog indicated that there is a significant variation in the number of zinc

finger motifs associated with C2H2-ZNF genes in these mammalian species. Noticeably,

the C2H2-ZNF genes from dog were found to encode a higher average of zinc finger motifs

as compared to the other mammals studied. It is possible that an increase in the number of

fingers within genes could confer advantageous additional functionality to the C2H2-ZNf

genes through a diversification ofthe possible nucleic acid and protein interactions.

We also observed a variation in the presence of N-terminal effector motifs, such as

SCAN or KRAB among orthologs, accounted by either gain or loss of these motifs.

However, loss of N-terminal effector domains by sequence degeneration was confirmed in

several cases in our study. A thorough analysis of the exon-intron structure of C2H2-ZNF

genes indicated a typical conserved exon-intron organization for C2H2-ZNF genes

associated with a SCAN-KRAB. Based on these observations, we propose a model of

evolution of C2H2-ZNF sub-families involving independent gain events of a SCAN and

KRAB domain each by an exon-shuffling mechanism and subsequent gene duplications

and loss of effector motifs by deletion or degeneration of the SCAN and/or KRAB domain.

152

3.5 Bïrth ami Deatli model of evolution

It was suggested from a study of a few chromosome 19 C2H2-ZNF clusters that

C2H2-ZNF genes evolve by positive selection (Schmidt and Durrett 2004). Our resuits and

analyses of the human C2H2-ZNF clusters and their syntenic counterparts in other

mammals suggests a “Birth and Death” model of evolution similar to that proposed by Nei

and coli. (Nei, Gu et al. 1997; Nei 2000) (See Figure 7B). According to this mode!, new

genes are created by duplication inc!uding tandem duplication and b!ock gene duplication

(birth). While some ofthem might acquire a new function and thus diverge functiona!ly, the

others may remain relatively unchanged in the genome for a long time. Again others

become pseudogenes following deleterious mutations or get de!eted from the genome

(death through inactivation or elimination). Though functiona! information is known for

on!y a handful of C2H2-ZNF proteins, the variations in the numbers C2H2-ZNF genes that

we found throughout the evolution of mamma!s and our phy!ogenetic analysis points to

duplication and loss as a guiding force in the evolution ofthese genes.

153

Thue

Ancestralspccics

Figure 7: Birth-and-death model of evolution.

The figure shows the two models associated with the evolution of multigene farnilies.

Open circles represent functional genes and closed circles represent pseudogenes.

(A) In concerted evolution, related genes belonging to the ancestral species evolve in a

concerted manner rather than independently in both Species 1 and Species 2.

(B) Birth and Death Mode! of evolution, where the genes evolve differently by duplication,

few are maintained in the genome for longer, while the others are deleted or become

pseudogenes.

Speces I Specics 2 Species E Species 2

fA) Ccrncerted evtiIutkr (13) Biith-and-death mx1e1of evolutior

154

3.6 C2H2-ZNF gene family: An analogy with the Olfactory Receptor

gene family

The olfactory receptor genes constitute the largest mammalian gene family with

more than 1000 members in human. However, 60% of these genes are pseudogenes. In

contrast to this, the olfactory receptor gene family in mouse comprises of roughly the same

number of genes as human, though the number of pseudogenes is only 20% (Glusman,

Yanai et al. 2001; Niimura and Nei 2003; Niimura and Nei 2005). Comparative analyses of

this gene family in human, mouse and non-human primates have revealed that differential

expansion and loss have guided the evolution of this gene family (Sharon, Glusman et al.

1999; Lapidot, Pupe! et al. 2001). However, human counterparts have accumulated a lot of

mutations, leading to the numerous pseudogenes in comparison to mouse or any other non

human primate. This is associated with the reduced chemosensory capacity in humans.

The olfactory receptor and the C2H2-ZNF gene families show similar pattems of

evolution. Differential gene expansion and loss have played an important role in the

evolution of both gene families in mammals. However, in contrast to the olfactory receptor

genes, C2H2-ZNF genes apparently do not accumulate pseudogenes in humans irrespective

of their large number. Studies on human C2H2-ZNF clusters have indicated that these

genes are rapidly evolving through positive selection and may acquire new functions after

duplication (Schmidt and Durrett 2004; Huntley, Baggott et al. 2006).

By making a correlation between the number of olfactory genes in human and

mouse and their functions in the respective organisms, we do understand that a reduced

chemosensory dependence in primates and non-human primates as compared to mouse can

155

be responsible for the large number of pseudogenes in human. Presently, the lack of large

scale analysis of the expression profile and ftmction of C2H2-ZNF genes preclude the

establishment of such type of conclusions. The extremely high number of hurnan C2H2-

ZNf genes, the species-specific expansions and loss in mammals leading to differential

evolution within primates and rodents, when put in perspective with functional information

ofthese proteins could give us interesting insights into the evolution ofthis gene family.

3.7 A few concerns to the study

We must acknowledge here three possible sources ofbias in our study.

First, errors in reporting the number of genes due to improper sequencing and

annotation in the available databases could be a primary source of bias to our study.

However, we did only consider genomes like human, chimpanzee, mouse, rat and dog

which are >94% complete to minimize significantly this source ofbias.

A second concern is that the loss of C2H2-ZNf clusters or genes that we see among

the species could be due to the fact that the genes were dispersed onto different

chromosomes due to translocation. Though we do accept this as a possibility, we conduct

an extensive analysis to rule out this possibility for the clusters we smdied in depth. For

example, in group I of the phylogenetic tree (Figure 5; Article) of the human cluster 19.12

and its syntenic clusters in chimpanzee, mouse, rat and dog, we observe three orthologs

(hZNF331, pZNF33Y and cZNF331) from human, chimpanzee and dog. There is no rodent

counterpart for these genes, which suggests a Ioss in rodents. To rule out the possibility that

these genes were dispersed onto other regions of the genome by transiocation, we conduct

156

an extensive homology TBLASTN search of the mouse and rat genomes using each of the

three orthoÏogs from human, chirnpanzee and dog as a query. for the three queries, the top

most blast hit was Zfpl4 from mouse and L0C97 124 from rat. We included these two

sequences into the dataset used for the phylogenetic analysis (see Methods; Article) of

cluster 19.12 in human and its syntenic counterparts in chimpanzee, mouse, rat and dog.

The phylogenetic tree revealed that the two mouse and rat sequences group with the three

orthoÏogs from human, chimpanzee and dog in group I. However, a doser look at the

sequence similarity between these sequences (< 60%) suggests that they cannot be

orthologs and the grouping we see could possibly be because ofthe fact that they were the

closest to the query sequences used

The third and final concem is that considering the extremely large numbers of

C2H2-ZNF in the human genome, we cannot rule out the possibility of pseudogenes.

Though we do not conduct an extensive search to look for possible pseudogenes, an

analysis of the open reading frames of the C2H2-ZNF genes considered in this study with

their translated sequences suggest that most of them are rnost likely flot pseudogenes. A

distribution curve (Figure 8) of the amino acid sequence length of the various C2H2-ZNF

genes shows that almost ail of the sequences have large open reading frames potentially

translated and functional.

157

160

—e-— Noof C2H2-ZNF

Figure 8: Plot ofthe amino acid sequence iengths of ail the C2H2-ZNF in the human

genome

140

120

100

80-

60

40

20

o - -- — - -- ——

Length of the C2H2-ZNF amino acid sequence

158

3.8 Merits of the study

Our study provides a comprehensive insight into the evolution of C2H2-ZNF

throughout several mammalian genomes. To summarize, the merits of our study are as

follows.

• A good range of species, with completely sequenced genomes was considered to

analyze the evolution of C2H2-ZNF genes in mammals.

• A stringent phylogenetic analysis of the syntenic clusters in human, chimpanzee,

mouse, rat and dog was performed using both maximum likelihood (RAxML)

and bayesian (Mr.Bayes) methods. Noticeably, unlike other studies on C2H2-

ZNF which use Xfin as an outgroup (Looman, Abrink et al. 2002; Shannon,

Hamilton et al. 2003; Huntley, Baggott et al. 2006), we conduct an extensive

search to include chicken (Gallus gallus) homologs in addition to Xfin as an

outgroup. The chicken sequences are considerably doser as an outgroup to the

species studied (human, chimpanzee, mouse, rat and dog).

The phylogenetic relationships we observed between C2H2-ZNF genes in the

syntenic clusters from the different species was found consistent with the overail

picture of the number of genes in the species and with the physical maps of the

clusters.

• A model for the evolutionary relationship of SCAN, SCAN-KRAB and KRAB

C2H2-ZNF subfamilies is proposed providing a possible explanation for

previously unresolved questions in the field.

159

3.9 Perspectives

The following studies could be done as a future approacli. to what is already

known.

• The compilation of comprehensive catalogues of the C2H2-ZNF gene

repertoires in chimpanzee, mouse, rat and dog. The detailed comparison of

the organization and numbers of C2H2-ZNF in complete repertoires.

• The stringent phylogenetic analysis of these repertoires to gain insights into

the various detailed mechanisms, which have taken place during the

evolution ofthis gene family in mammals.

• The more detailed analysis of the physical mapping of genes within clusters

to gain insight into the molecular mechanisms involved in the expansion of

these genes. This could include the analysis of the possible repeated

sequences that are bordering these C2H2-ZNF and that may be involved in

this phenomenon, the analysis of the orientation, distances between genes

and exon-intron organisation.

• The more comprehensive study of pseudogenes

Clearly, more detailed bio-informatics and functional studies are stiil required for a better

understanding of the driving force behind the expansion of C2H2-ZNF genes in mammals.

160

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