Characterisation of GATA3 mutations in the Hypoparathyroidism ...

GATA3 mutations in HDR syndrome

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Characterisation of GATA3 mutations in the Hypoparathyroidism,

Deafness and Renal Dysplasia (HDR) Syndrome

M. Andrew Nesbit1, Michael R. Bowl1, Brian Harding1, Asif Ali1, Alejandro Ayala2, Carol

Crowe3, Angus Dobbie4, Geeta Hampson5, Ian Holdaway6, Michael A. Levine7, Robert

McWilliams8, Susan Rigden9, Julian Sampson1 0, Andrew Williams1 1, and Rajesh V.

Thakker1

1Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford,

Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford,

United Kingdom,2Pediatric and Reproductive Endocrinology Branch, National Institutes of Health, Bethesda,

Maryland, USA,3Division of Genetics, Department of Pediatrics, MetroHealth Medical Center, 2500

MetroHealth Drive, Cleveland, Ohio, USA,4Department of Clinical Genetics, The Churchill Hospital, Oxford, United Kingdom,5Department of Chemical Pathology, The Guy's, King's College and St Thomas' Hospitals

Medical and Dental School, St Thomas' Hospital, London, United Kingdom,6Department of Endocrinology, Auckland Hospital, Park Road, Auckland 1, New Zealand,

7Department of Pediatric Endocrinology, The Children’s Hospital at The Cleveland Clinic,

Cleveland, Ohio, USA,8Divisions of Hematology and Oncology, Mayo Clinic, Rochester, Minnesota, USA,9Paediatric Renal Unit, Guy's Hospital, St Thomas Street, London, United Kingdom,1 0Institute of Medical Genetics, University of Wales College of Medicine, Cardiff, United

Kingdom,1 1Department of Nephrology, Morrison Hospital, Swansea, United Kingdom.

JBC Papers in Press. Published on February 24, 2004 as Manuscript M401797200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on March 23, 2018

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Address correspondence to: Professor R.V. Thakker, Academic Endocrine Unit, Nuffield

Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology

and Metabolism, Churchill Hospital, Oxford, OX3 7LJ, United Kingdom.

Phone: 44-1865-857501; FAX: 44-1865-857502; E-mail: [email protected].

M. Andrew Nesbit and Michael R. Bowl contributed equally to this work.

Running Title: GATA3 mutations in HDR syndrome


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SUMMARY

The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal

dominant disorder caused by mutations of the dual zinc-finger transcription factor, GATA3.

The C-terminal zinc finger (ZnF2) binds DNA, whilst the N-terminal finger (ZnF1)

stabilizes this DNA binding and interacts with other zinc finger proteins, such as the

Friends of GATA (FOG). We have investigated 7 HDR probands and their families for

GATA3 abnormalities and have identified 2 nonsense mutations (Glu228Stop and

Arg367Stop); 2 intragenic deletions that result in frameshifts from codons 201 and 355

with premature terminations at codons 205 and 370, respectively; 1 acceptor splice site

mutation that leads to a frameshift from codon 351 and a premature termination at codon

367; and 2 missense mutations (Cys318Arg and Asn320Lys). The functional effects of

these mutations, together with a previously reported GATA3 ZnF1 mutation and 7 other

engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation,

yeast two-hybrid and glutathione-S-transferase pull-down assays. Mutations involving

GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of

ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding

affinity. These findings are consistent with the proposed 3-dimensional model of ZnF1,

which has separate DNA and protein binding surfaces. Thus, our results, which expand the

spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site

mutation, help to elucidate the molecular mechanisms that alter the function of this zinc-

finger transcription factor and its role in causing this developmental anomaly.

Keywords: congenital anomaly, zinc finger, transcription


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INTRODUCTION

GATA3 belongs to a family of zinc-finger transcription factors that are involved in

vertebrate embryonic development (1-3). The six mammalian GATA proteins (GATA-1 to -

6) share related CysX2CysX1 7CysX2Cys (where X represents any amino acid residue) zinc

finger DNA-binding domains (Figure 1) and bind to the consensus motif 5’-A/T-GATA-

A/G-3’(4). The carboxy-terminal finger (ZnF2)1 is essential for DNA binding, whereas

the amino-terminal finger (ZnF1) appears to stabilize this binding and to physically interact

with other multi-type zinc finger proteins, such as the Friends of GATA (FOG) (5-7).

Thus, FOG-1 and FOG-2 have been shown, in mammals, to modulate the biological

activities of GATA1 and GATA4, respectively (5-7). Furthermore, the importance of these

interactions of GATA and FOG family members are underscored by their evolutionary

conservation, as it has been shown that the Drosophila GATA factor, Pannier, interacts with

a FOG-like protein referred to as U-shaped (Ush) (8,9). The mammalian GATA factors can

be subdivided into two families based on their structures and patterns of expression (10,11).

Thus, the structurally related proteins GATA4, -5, and -6 are expressed in overlapping

patterns in the heart, gut, urogenital system, and smooth muscle cell lineages, whilst GATA1,

-2, and -3 are expressed in the hematopoietic cell lineages in which they control

development of the erythroid, hematopoietic stem cell and T cell lineages, respectively

(10,11). In addition, GATA3 is also expressed in the developing parathyroids, inner ear and

kidneys (12,13). These expression patterns are consistent with the disease phenotypes that

have been reported in the few patients with genetic abnormalities involving three of the

GATA members. Thus, GATA1 mutations lead to dyserythropoietic anemia,

thrombocytopenia (14) and the megakaryoblastic leukemia of Down’s syndrome (15);

GATA3 haploinsufficiency is associated with the hypoparathyroidism, deafness and renal

1 The abbreviations used are: ZnF, zinc finger; HDR, hypoparathyroidism, deafness andrenal dysplasia; FOG, friend of GATA; Ush, U shaped; GST, glutathione-S-transferase;EMSA, electrophoretic mobility shift assay; WT, wildtype; PTH, parathyroid hormone;PEG, polyethylene glycol; TA, transactivating; CAPS, 3[cyclohexylamino]-1-propanesulphonic acid; GFP, green fluorescent protein.


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dysplasia (HDR) syndrome (16); and GATA4 hemizygosity has been observed in some

patients with congenital heart disease (17). More than 90% of HDR syndrome patients

have hypoparathyroidism and deafness, and more than 80% have renal tract abnormalities

(16,18,19). The hypoparathyroidism is characterised by symptomatic or asymptomatic

hypocalcemia with undetectable or inappropriately normal serum concentrations of

parathyroid hormone (PTH), and normal brisk increases in plasma cAMP in response to

PTH infusion, which indicates normal sensitivity of the PTH receptor (18). The

sensorineural deafness is usually bilateral although the hearing loss may vary in its severity

(18,20-22). The renal tract abnormalities, which may be uni- or bi-lateral, consist of: renal

cysts that may cause pelvicalyceal deformities and/or compression of the glomeruli and

tubules that may lead to kidney failure; renal aplasia or hypoplasia; and vesicoureteral reflux

(16,18-22). The precise manner in which GATA3 mutations cause these congenital

abnormalities of the parathyroids, inner ear and kidneys remains to be elucidated. In order

to gain further insights into the structure-function relationships of GATA3, we have studied

additional HDR patients for GATA3 abnormalities, and have investigated the effects of

GATA3 mutations on DNA binding and protein interactions.


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EXPERIMENTAL PROCEDURES

Patients - Ten patients with HDR from 7 unrelated families were ascertained (Table I).

Four families (19/1992, 8/2000, 2/2001, and 13/2001) were from Northern Europe, two

families (19/2000 and 16/2001) were from North America, and one family (16/1998) was

from Samoa. All 10 patients had hypoparathyroidism with serum calcium ranging from

1.01 to 2.00 mmol/l and this was associated with tetany or seizures in 4 patients, but was

asymptomatic in 6 patients (Table I). Bilateral sensorineural deafness was found in all 10

patients with the age at diagnosis ranging from <1 to <30 years. Renal abnormalities were

found in 7 patients, of which 2 patients had developed end-stage renal failure, 2 had

hypoplastic kidneys, and another 2 had agenesis of the right kidney.

DNA sequence analysis of the GATA3 gene - Venous blood was obtained after informed

consent, as approved by the local ethical committee, and used to extract leukocyte DNA

(23). Nine pairs of GATA3-specific primers were used for the PCR amplification of the six

exons and ten intron-exon boundaries (Figure 1) utilising 150ng genomic DNA as

described (24). The DNA sequences of both strands were determined by Taq polymerase

cycle sequencing (24) and resolved on a semi-automated detection system (373 sequencer

Applied Biosystems, Foster City, CA). DNA sequence abnormalities in the probands,

which were confirmed either by restriction endonuclease analysis (24), or by allele specific

oligonucleotide (ASO) hybridisation (25), or by a modified version of the amplification

refraction mutation system (ARMS) (26), were demonstrated to cosegregate with the

disorder and to be absent in the DNA obtained from 55 unrelated individuals.

Electrophoretic mobility shift assays (EMSAs) - COS-1 cells, which do not

endogenously express GATA3, were transfected using Lipofectamine Plus (Invitrogen,

Carlsbad, CA) with either a wild type GATA3 construct prepared in pcDNA 3.1 (GATA3-

pcDNA3) (Invitrogen, Carlsbad, CA) or a construct harbouring the mutation that was

introduced by the use of site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA)

(16). Forty-eight hours post-transfection, the cells were harvested and nuclear extracts


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prepared for use in binding reactions that utilised a 3 2P-labelled double-stranded

oligonucleotide containing the GATA3 consensus as described (16). The binding reactions

were resolved by non-denaturing 6% polyacrylamide gel electrophoresis (PAGE). Western

blot analysis using HG3-31 monoclonal antibody against GATA3 (Santa Cruz

Biotechnology, Inc. Santa Cruz, CA) was used to detect the presence of GATA3 protein in

the nuclear extracts (16). For dissociation shift assays (14,27), unlabelled competitor DNA

was added to an 100-fold excess to the binding reactions, and aliquots removed after 0, 10,

30, and 60 minutes for non-denaturing PAGE.

Nuclear localization studies using GATA3-green fluorescent protein (GFP) fusion

constructs – The wild type and mutant GATA3 constructs were subcloned in-frame into the

mammalian expression vector pEGFP-C1 (BD Biosciences Clontech, Palo Alto, CA) as

previously described (28). COS-1 cells were transfected with the GATA3-GFP constructs,

using Lipofectamine Plus (Invitrogen, Carlsbad, CA), and after 24 hours the cells were

replated at lower density onto 70% ethanol-treated coverslips and cultured for a further 24

hours. The cells were then washed with phosphate-buffered saline (PBS), fixed with

freshly prepared 4% paraformaldehyde/PBS for 30 minutes, washed with PBS, and

mounted with 4’, 6-diamidino-2-phenylindole (DAPI)-containing Vectashield (Vector

Laboratories, Burlingame, CA), as described (28). The DAPI /GFP images were visualised

using a Nikon Eclipse E400 microscope with a Y-FL Epi-fluorescence attachment and a

triband DAPI-FITC-Rhodamine filter (28).

GATA3 minigene construct for mRNA splicing studies - A minigene containing GATA3

exons 4, 5, and 6 was constructed. Each exon was PCR amplified from genomic DNA

using exon-specific primers and conditions that were utilised for DNA sequence analysis

(16). The PCR products were cloned directly into pGEM-T (Promega, Madison WI) and

sequenced to determine orientation and absence of Taq introduced secondary mutations.

Each exon was excised from pGEM-T using appropriate restriction endonucleases and

directionally subcloned in a four-way ligation reaction into pcDNA 3.1. COS-1 cells were


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transfected with the plasmid, as described above, and after 48 hours the cells were harvested

and RNA prepared (24) for use in reverse-transcription PCR (RT-PCR) that utilised AMV

reverse-transcriptase (Life Sciences Inc. St. Petersburg, FL) and a reverse pcDNA3.1

primer to synthesize the first-strand cDNA. A control reaction without reverse transcriptase

was also performed. The PCR reaction contained 1.5mM MgCl2, 250mM dNTP

(Invitrogen, Carlsbad, CA), 0.3mM of each primer (Forward exon primer 5 5'-

TCTGCAATGCCTGTGGGCTCTAC-3', and reverse exon 6 primer 5'-

CTAACCCATGGCGGTGACCATGC-3'), and 1U Taq DNA polymerase (Invitrogen,

Carlsbad, CA) in 50m l standard PCR buffer (16). Amplification conditions were,

denaturation at 95ºC for 5 min, followed by 30 cycles of 94ºC for 15s, 65ºC for 15s, and

72ºC for 1 min, followed by final extension at 72ºC for 5 min and rapid cooling to 20ºC.

Yeast Two-hybrid assays - In vivo interactions between the GATA3 N-terminal zinc

finger (ZnF1) (Figure 1) and FOG2 ZnFs 1, 5, 6, and 8 were studied using a yeast two-

hybrid system (BD Biosciences Clontech, Palo Alto, CA) (29). GATA3 ZnF1 (amino acids

261-293) was generated by cloning a PCR product, amplified from the wild type GATA3

expression construct (16), in-frame, into the Gal4 DNA-binding domain (BD)-encoding

plasmid, pGBKT7 (30). Mutations were introduced into this construct by site-directed

mutagenesis (QuikChange, Stratagene, La Jolla, CA). FOG2 ZnFs were generated by RT-

PCR using Human Embryonic Kidney (HEK) 293 cell RNA as template. Each FOG2 ZnF

(ZnF1 amino acids 236-290; ZnF5 amino acids 531-617; ZnF6 amino acids 661-747; and

ZnF8 amino acids 1100-1151) was cloned in-frame into the Gal4 activation domain (AD)-

encoding plasmid, pGADT7. The p53-pGBKT7 and Large T antigen-pGADT7 plasmids

(BD Biosciences Clontech, Palo Alto, CA) were used as controls (31,32). Competent

AH109 yeast cells were transformed sequentially with the appropriate GATA3 and FOG2

ZnF plasmid constructs using the LiAc/SS-DNA/PEG procedure (33). The transformants

were selected on Leu-Trp- (double drop-out, DDO) minimal media plates by growth at 30ºC

for 3 days. Transformants were then patched onto His-Ade-Leu-Trp- (quaternary drop out,


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QDO) media plates and monitored for growth for up to 3 days. Expression of GATA3 and

FOG2 Gal4 fusion proteins was confirmed by preparing protein extracts from each clone

according to the manufacturer’s instructions (BD Biosciences Clontech, Palo Alto, CA) and

analyzing them by SDS-PAGE in Tris-Glycine-SDS buffer (Biorad, Hercules, CA) and

electro-transference onto PolyScreen PVDF transfer membrane (NEN Life Science

Products, Inc. Boston, MA) in CAPS buffer (10mM 3-[cyclohexylamino]-1-

propanesulphonic acid pH11 (Sigma Chemical Co. St. Louis, MO)). Western blot analysis

was performed with antibodies to either the Gal4-AD (FOG2-pGADT7 constructs) or the

Gal4 DNA-BD (GATA3-pGBKT7 constructs), according to the manufacturers

instructions (BD Biosciences Clontech, Palo Alto, CA) except that Gal4 DNA-BD antibody

was used at 50ng/ml and Gal4-AD antibody at 100ng/ml (29). A secondary antibody, goat

anti-mouse horseradish peroxidase, HRP (Biorad, Hercules, CA) was used at 1/5000 and

detected by using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia

Biotech, Piscataway, NJ).

Glutathione-S-transferase fusion proteins and pull-down assays - The glutathione-S-

transferase (GST) fusion proteins contained FOG2 ZnF1, ZnF5, ZnF6, and ZnF8 fused

downstream of the GST protein in the vector pGEX-4T-1 (Amersham Pharmacia Biotech,

Piscataway, NJ). The expression of GST fusion proteins was carried out in Escherichia coli

BL21 (34). 3 5S-labelled wild-type or mutant GATA3 proteins were prepared by in vitro

transcription/translation (TNT system, Promega, Madison WI) using GATA3-pcDNA3 or

constructs harbouring selected mutations, and aliquots utilized to monitor 3 5S-methionine

incorporation by SDS-PAGE(16). In vitro binding assays using 1mg of the fusion protein

attached to glutathione sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) and

1ml of the radiolabelled GATA3 protein were performed in 300ml of binding buffer

(150mM NaCl, 20 mM Tris-HCL pH 7.5, 0.1% Igepal CA-630, 20mM ZnSO4, 0.25%

bovine serum albumin, 1mM b-mercaptoethanol, 1.5mM phenylmethylsulphonyl fluoride)

and incubated with mixing for 1h at 4ºC (35). The glutathione sepharose 4B/FOG2 fusion


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protein/GATA3 complexes were recovered by centrifugation (20,000 x g, 2 minutes) and

washed 4 times with 450ml cold binding buffer. The proteins were released by boiling in

15ml Laemmli sample buffer (Biorad, Hercules FL) and analyzed by SDS-PAGE (12%

polyacrylamide resolving gel in Tris/Glycine/SDS running buffer (Biorad, Hercules, FL).

The gel was fixed, and then soaked in Amplify (Amersham Pharmacia Biotech, Piscataway,

NJ), prior to autoradiography (36).

Computer modeling of GATA3 ZnF1 structure - The three-dimensional structure of the

murine GATA1 N-terminal zinc finger has been reported (37), and as the N-terminal zinc

fingers of GATA1 and GATA3 are over 90% identical, we modeled the position of the

GATA3 mutants on this framework. The GATA1 ZnF1 three-dimensional structure is

archived in the Protein Data Bank (PDB) at the European Bioinformatics Institute (EBI)

with the accession number 1GNF (http://oca.ebi.ac.uk/oca-bin/ccpeek?id=1GNF) and was

visualized using the MDL Chime program (MDL Information Systems, Inc., San Leandro,

CA)


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RESULTS

Mutations in HDR families - DNA sequence analysis of the entire 1332bp coding region

together with the associated splice sites and 5’ and 3’ untranslated regions (UTRs) of the

GATA3 gene from each of the seven probands with HDR revealed the presence of seven

heterozygous mutations (Figure 1, Table II), six of which were novel and one of which had

previously been reported in an unrelated Japanese family (22). Thus, two of the mutations

were nonsense mutations (Figure 2), two were frameshifting deletions, two were missense

mutations, and one was an acceptor splice site mutation (Figure 3). The occurrence of the

nonsense, frameshifting deletions and missense mutations in the probands was confirmed

either by restriction enzyme analysis (Figure 2), or by ASO hybridisation analysis, or by

ARMS (Table II). The acceptor splice site mutation, which involved a g to t transversion of

the invariant ag (Figure 3) was confirmed by repeat DNA sequence analysis on

independently obtained PCR products. This predicted a loss of this acceptor splice site and

the possible use of another naturally occurring, but normally unused acceptor splice site at

codons 351 to 353 (Figure 3). These predicted effects on mRNA splicing were assessed by

expressing wild-type and mutant GATA3 minigene constructs that encompassed exons 4, 5

and 6, in COS-1 cells. This revealed utilisation of the alternative acceptor splice site that

would lead to a loss of 8 nucleotides from the mRNA. This resulted in a frameshift which,

if translated, would produce a missense peptide with a premature termination at codon 367.

Cosegregation of the GATA3 mutations and HDR was demonstrated in the available

members from families 8/2000 (Figure 2), 9/2000, and 2/2001, whilst in the probands from

families 13/2001 and 16/2001, the mutations were demonstrated to be absent in the parents

and hence were arising de novo (Table II). In addition, the absence of these DNA sequence

abnormalities in 110 alleles from 55 unrelated normal individuals indicated that these

abnormalities were mutations and not functionally neutral polymorphisms that would be

expected to occur in >1% of the population. All of the seven mutations, which occurred in

exons 3, 4, 5, or 6 (Figure 1), predict structurally significant changes (Table II). Thus, the


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E228X (Glu228Stop) and frameshift deletion occurring in codon 201 are predicted, if

translated, to lead to truncated GATA3 proteins that lack both ZnFs; the R367X

(Arg367Stop), the frameshift deletions occurring in codon 355, and the acceptor splice site

mutation at the intron 5/exon 6 boundary are predicted to lead to truncated GATA3 proteins

that lack the C-terminal region adjacent to ZnF2, and the missense mutations C318R

(Cys318Arg) and N320K (Asn320Lys) are predicted to disrupt ZnF2 of GATA3 (Figure

1). The effects of these mutations together with the W275R (Trp275Arg) that was reported

in a Japanese HDR patient (22) were further assessed in DNA binding studies. The effects

of the acceptor splice site mutation found in family 16/2001 (Figure 3) were not assessed

separately as the predicted protein is almost identical to that resulting from the frameshift

deletion found in family 16/1998 (Figure 1, Table II).

DNA binding and subcellular localisation studies - All of the HDR associated GATA3

mutations, with the exception of one, W275R, are predicted to disrupt ZnF2 or its adjacent

C-terminal region (Figure 1), and the results of Western blot analysis are consistent with

this (Figure 4). ZnF2, which is the C-terminal zinc finger, is essential for DNA binding and

thus all of these HDR associated GATA3 mutations would predict a disruption of DNA

binding (Table II). However, the W275R mutation lies within ZnF1 and its effects are more

difficult to predict although some naturally occurring and some engineered GATA1 mutants

of the N-terminal zinc finger, ZnF1, have been shown to destabilize DNA binding or

protein-protein interactions (14,35,38-40). We therefore engineered the equivalent 7

GATA3 mutants, E263V (Glu263Val), C264R (Cys264Arg), GA268/269QT

(GlyAla268/269GlnThr), P273T (Pro273Thr), R276Q (Arg276Gln), D278G (Asp278Gly)

and D278Y (Asp278Tyr), so as to facilitate a more comprehensive study of the 25 residues

forming the GATA3 ZnF1 (Figure 1). These residues were selected for engineering

mutants as they either are the non-conserved residues of ZnF1 when compared to their

respective ZnF2 counterpart, or they are the equivalent counterparts to GATA1 disease-

causing mutations (38-40). We assessed these GATA3 mutants (i.e. the ones associated


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with HDR (Figure 1) and the 7 engineered GATA3 ZnF1 mutations) initially for altered

DNA binding by EMSAs (Figure 4), using nuclear extracts from COS-1 cells transfected

with either the wild-type or mutant GATA3 constructs. In addition, an assessment of the

subcellular localisation of the GATA3 mutants, using GATA3-GFP constructs, was also

undertaken and this revealed that 12 of the mutants, which retained ZnF1 (Figure1),

accumulated in the nucleus and were indistinguishable from the WT-GATA3 (Figure 4).

However, the 2 mutants (deletion of C in codon 201 and E228X) that lacked ZnF1 did not

accumulate in the nucleus. These findings are consistent with the nuclear localisation signal

for GATA3 being contained within residues 249 to 311 that encompass ZnF1 (41). The

EMSA studies revealed that the GATA3 mutants which disrupted or lead to a loss of ZnF2

(Figure 1 and Table II), all resulted in a loss of DNA binding. Furthermore, addition of a

two-fold excess of these mutant GATA3 nuclear extracts to the wild type, did not

significantly alter binding by WT-GATA3 (data not shown), thereby suggesting an absence

of a dominant-negative effect due to heteroduplex formation. This is consistent with the

development of an HDR phenotype in patients who have haploinsufficiency due to a

deletion of the GATA3 gene (16). The GATA3 mutants involving ZnF1, all retained DNA

binding (Figure 4). However, these ZnF1 mutants differed in the stability of their binding

to DNA which resulted in altered rates of dissociation. Thus, the HDR associated mutant

W275R and the engineered mutants GA268/269QT, D278G and D278Y had dissociation

rates similar to that of the wild-type GATA3 (Figure 4), whereas the engineered mutants

E263V, C264R, P273T and R276Q had a more rapid rate of dissociation (Figure 4). These

results indicate that the ZnF1 GATA3 residues E263, C264, P273 and R276 are critical for

stabilising the DNA binding by ZnF2 and that this is likely to involve interactions with other

multi-type zinc finger proteins, in a manner similar to that reported for GATA1 ZnF1

(14,35,38-40). For example, the engineered GATA1 mutant C204R, which is equivalent to

the GATA3 C264R, has been reported to destabilize DNA binding (14) and to abolish the

interaction with FOG ZnF6 (35). However, the HDR GATA3 mutant W275R and the


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engineered mutants GA268/269QT, D278G and D278Y did not alter the stability of the

DNA binding (Figure 4) and to further elucidate the role of these residues and their

mutations, we utilised a yeast two-hybrid assay.

Yeast two-hybrid assay - GATA1 ZnF1 and GATA4 ZnF1 interact with the zinc finger

proteins FOG1 and FOG2, respectively (5-7). We investigated FOG2 for interactions with

GATA3, because of their similar temporo-spatial expression patterns (6,12). Thus, in

mouse embryos older than 11.5 days both GATA3 and FOG2 are expressed in the same

tissues that include the otic vesicle and the developing kidney (6,12). In addition FOG2 has

been shown to interact with GATA3 in mouse embryos (7). These interactions between

GATA factors and the FOG proteins involve the GATA ZnF1 and several of the zinc

fingers of the FOG protein. For example, the GATA1 ZnF1 interacts with 4 of the nine

zinc fingers (-1, –5, -6 and –9) of FOG, and 4 of the eight zinc fingers (-1, -5, -6, and –8) of

FOG2 (42). We selected to investigate the 4 involved zinc fingers (-1, -5, -6 and –8) of

FOG2 for interactions with wild-type and mutant GATA3 ZnF1 in a yeast two-hybrid

assay. One GATA3 construct and one FOG2 construct were sequentially transformed into

the yeast reporter strain AH109, and yeast containing both plasmids were selected on

minimal DDO medium that lacked leucine and tryptophan (Figure 5a). Co-expression of

the GATA3 and FOG2 Gal4 fusion proteins was confirmed by Western blotting of yeast

protein extracts, prepared from each clone, and detected using antibodies against either the

Gal4 DNA-BD or the Gal4-AD (data not shown). These yeast colonies were then patched

onto minimal QDO medium that lacked leucine, tryptophan, histidine and adenine to select

for those yeast in which a protein-protein interaction had occurred (Figure 5b). Interaction

between the GATA3 and FOG2 zinc fingers would bring the Gal4 DNA-BD into close

juxtaposition with the AD at the reporter gene promoter, thereby enabling transcription of

the reporter gene. Disruption of this interaction by the GATA3 mutant would lead to a loss

of expression of the reporter genes. The results revealed interactions between the wild-type

GATA3 ZnF1 and each of the four FOG2 zinc fingers (-1, -5, -6 and –8) (Figure 5).


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However, the GATA3 mutants C264R, E263V and GA268/269QT did not interact with any

of the FOG2 zinc fingers as evidenced by an absence of yeast growth. The GATA3 mutant

W275R similarly abolished interaction with FOG2 zinc fingers 1, 5 and 8, but retained

interaction with ZnF6, whilst the D278G and D278Y mutants retained interaction with all

FOG2 zinc fingers with the exception of ZnF8. However, the P273T and R276Q mutants

retained interaction with all four FOG2 zinc fingers, thereby suggesting that they exert their

effect solely by loss of DNA-binding stabilization. These results of the yeast two-hybrid

assay were confirmed by GST pull-down assays.

GST pull-down assays - GST pull-down assays were performed using full-length

GATA3 expressed in a rabbit reticulocyte system, and FOG2 ZnF-GST fusion proteins.

The wildtype GATA3, and the P273T and R276Q mutants were retained by FOG2 ZnFs 1,

5, 6, and 8, whereas the W275R mutant was retained only with FOG2 ZnF6 (Figure 5c, data

shown for wildtype and W275R). In contrast, the E263V, C264R, and GA268/269QT

mutants were not retained by any of the four FOG2 ZnFs (Figure 5c, data shown for

C264R), whilst the D278G and D278Y mutants were retained by FOG2 ZnF 1, 5, and 6 but

not FOG2 ZnF8. These GST pull-down results, which confirm the results of the yeast two-

hybrid assay, are in agreement with those previously reported for interactions between

GATA1 and FOG2 ZnFs (42).


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DISCUSSION

Our results, which have identified seven mutations of the GATA3 gene in seven

HDR probands and their families (Table II), expand the spectrum of mutations, report the

first acceptor splice site mutation (Figure 3), and further establish the role of GATA3

haploinsufficiency in the etiology of this developmental disorder. In addition, our studies of

these GATA3 mutations help to increase our understanding of the underlying DNA binding

and protein interactions that are involved for the function of this zinc finger transcription

factor. Thus, all the mutations that disrupt either ZnF2 or the basic amino acids located C-

terminal to it, lead to a loss of DNA binding (Figure 4), whilst those that disrupt ZnF1 do

not lead to a loss of DNA binding but instead alter interactions with FOG2 (Figure 5)

and/or change DNA binding affinity (Figure 4). For example, the two missense mutations,

C318R and N320K (Figure 1 and Table II), which result in alterations of evolutionarily

conserved residues in ZnF2s of the GATA family members, are predicted to disrupt the

tertiary structure either directly or via a loss of co-ordination of the zinc ion. This in turn

results in a loss of DNA binding and hence a likely alteration in the transcription of target

genes. Similarly, the 3 mutations (2 frameshifts starting at codons 351 and 355, and the

nonsense mutation R367X) involving the residues on the C-terminal side of ZnF2 (Figure 1

and Table II) also result in a loss of DNA binding (Figure 4). These 3 mutations involve

codons 364-369, whose equivalents in GATA1 have been shown to be essential for DNA

binding, either by direct contact with DNA or by stabilization of nearby residues that contact

DNA (43).

In contrast to these GATA3 ZnF2 mutants, the 8 ZnF1 mutants (the HDR-

associated W275R and the 7 engineered mutants) all retained DNA binding activity (Figure

4). These findings for human GATA3 ZnF1 are consistent with those reported for the

chicken GATA3 ZnF1 (44) which has been shown to bind GATA or GATC motifs even in

the absence of ZnF2. Such studies (27,44,45) have indicated that GATA ZnF1 may serve to

stabilize the binding of ZnF2 to gene promoters or enhancers that contain double or


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palindromic GATA sites, and thereby help in distinguishing between genes that are

regulated by different GATA members. However, the GATA3 ZnF1 mutants in our study

did show differences in both their DNA binding affinities (Figure 4) and in interactions

with the 4 of the 8 FOG2 ZnFs that were studied (Figure 5). Thus, the mutants E263V and

C264R had low DNA binding affinities and a lack of interactions with FOG2 ZnF1, 5, 6,

and 8; the P273T and R276Q mutants had low DNA binding affinities but retained

interactions with the 4 FOG2 ZnFs; the W275R, D278G and D278Y had a normal DNA

binding affinity and interacted with some FOG2 ZnFs, e.g. W275R interacted with FOG2

ZnF6, and D278G and D278Y interacted with FOG2 ZnF1, 5 and 6; whilst GA268/269QT

had a normal DNA binding affinity but a lack of interactions with any of the 4 FOG2 ZnFs.

The altered DNA binding affinities observed with E263V and C264R but not

GA268/269QT, may be attributed to the disruption of the ZnF1 structure and a lack of zinc

ion co-ordination that is likely to result with the E263V and C264R mutants, but not the

GA268/269QT mutant that involves substitutions for residues that are present in equivalent

positions in ZnF2 (Figure 1). However, any further explanation for these results is difficult

to provide on the basis of the primary structure of GATA3 ZnF1, but an analysis of the

predicted 3-dimensional structure of ZnF1 (Figure 6) may be useful as it indicates that there

may be specific DNA and protein binding surfaces. Thus, E263, C264, G268 and A269 are

clustered to form a surface that is important for protein binding e.g. with FOG2 ZnFs;

whilst W275 and D278 reside on another surface that may be important for interactions

with ZnFs 1, 5, and 8, and ZnF8 respectively; whereas P273 and R276 reside on a different

surface that is involved in binding DNA but not FOG2 ZnFs.

The role of the HDR associated W275R mutation is of further interest in this

model. The W275R mutation is located amongst residues (P273 and R276) that form a

DNA binding surface (Figure 6) and yet it leads to a loss of protein interactions with FOG2

ZnFs 1, 5, and 8, and not an alteration in DNA binding affinity. This suggests a dual role

for the WRR peptide (codons 275 to 277), which is conserved in both ZnF1 and ZnF2


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(Figure 1), in binding to DNA as well as FOG2. These results are consistent with those

reported from studies of GATA4 ZnF2, in which the equivalent conserved residues were

mutated and shown to be critical for DNA binding and for interactions between GATA4

ZnF2 and the protein p300/CBP (46,47). Furthermore, a GATA1 ZnF1 mutant, which

involved the equivalent GATA3 residue R276, failed to bind GATA motifs but interacted

normally with FOG (40). All these observations indicate that the WRR peptide is involved

in separate FOG-GATA interacting and DNA binding functions, and a 3-dimensional

model of ZnF1 (Figure 6) is consistent with this if the aromatic side-chain of the W275

residue projects away from the DNA binding surface formed by P273, R276 and R277, and

is thereby available to interact with FOG2 ZnFs. We have concentrated on studying the

effects of GATA3 mutants on the interactions with FOG2 because of their similar temporo-

spatial expression patterns (6,7). However, GATA3 also interacts with other transcription

factors that include GATA1, GATA2 (48,49), Sma and Mad-related protein 3 (smad3) (50),

specificity protein 1 (SP1) (51), erythroid Krüppel-like factor (EKLF) (51), and rhombotin

2 (RBNT2) (52). GATA2 (53), smad3 (54) and RBNT2 (55) are expressed in kidney,

whilst SP1 is expressed in both kidney (56) and parathyroids (57) and thus, it may be

possible for HDR-associated GATA3 mutations to disrupt interactions with these proteins,

provided that they were expressed contemporaneously.

An examination of the HDR associated GATA3 mutations together with the

observed phenotypes does not establish a correlation (Tables I and II), and this is well

illustrated by the 2 unrelated families from Britain and Japan (22) who had an identical

R367X mutation but different phenotypes. Thus, the British patient 13/2001 (Table I and

Table II) had hypoparathyroidism and deafness, but no renal abnormalities, whilst both

Japanese patients had hypoparathyroidism and renal abnormalities but no deafness (22).

Furthermore, even within families with patients harboring identical GATA3 mutations there

appears to be a variable expression of renal abnormalities as illustrated by family 8/2000

(Figure 2, Table I). The basis of these phenotypic differences in patients with the same


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mutation remains to be elucidated. One possibility is that there may be different levels of

compensation by other GATA family members in different patients. This hypothesis seems

attractive, particularly as GATA2 and GATA3 have been shown to be able to partially

compensate for the loss of GATA1 in the differentiation of hematopoietic lineages, when

placed under the control of the GATA1 locus in transgenic mice (58,59). However, it is

important to note that studies of mice lacking GATA1 have demonstrated that GATA2 does

not compensate for the loss of GATA1 function in vivo (60), thereby indicating that

extrapolation of compensatory mechanisms to the native situation requires cautious

interpretation. Additional studies investigating for genotype-phenotype correlations in

HDR patients and for alterations in the expression of GATA family members that may

compensate for reduced GATA3 expression are required. In summary, our studies have

shown that HDR-associated GATA3 mutations may either disrupt DNA binding or protein

interactions with FOG2, and that these are consistent with the roles of the zinc finger

domains and with the proposed 3-dimensional model. However, the manner in which these

GATA3 mutations lead to parathyroid, otic vesicle and renal anomalies remains to be

elucidated.


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Acknowledgments

We are grateful to the Medical Research Council (UK) (M.A. Nesbit, M.R. Bowl, B.

Harding, A. Ali, and R.V. Thakker). M.R. Bowl is an M.R.C. PhD student and A. Ali is an

M.R.C. Clinical Training Fellow.


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Figure Legends

Figure 1

Schematic representation of the genomic structure of the GATA3 gene illustrating the

locations of mutations identified in HDR patients. The human GATA3 gene consists of 6

exons that span 20kb of genomic DNA and encode a 444 amino acid transcriptional factor

that contains two transactivating domains (TA1 and TA2) and two zinc fingers (ZnF1 and

ZnF2). The sizes of exons 1, 2, 3, 4, 5, and 6 are 188bp, 610bp, 537bp, 146bp, 126bp, and

806bp, respectively. The ATG (translation start) site is in exon 2 and the TAG (stop) site is

in exon 6. The locations of the 7 HDR mutations identified by the present study are shown

(numbers 1 to 7 which corresponds to mutations detailed in Table II) together with the 6

previously reported mutations (labelled a to f: a, R277X; b, R367X; c, deletion frameshift

(del,fs) from codon 156; d insertion frameshift (ins,fs) from codon 301; e, in-frame deletion

(del, inf) 316-319; f W275R (16,22). In addition, 6 whole gene deletions (del) have been

previously reported (16,22), yielding a total of 19 GATA3 abnormalities identified in HDR

patients. Nine of the 10 HDR mutations, which affect the region encompassing the two zinc

fingers and the adjacent C-terminal region, are further detailed above in the amino acid

sequence, in which every tenth amino acid is numbered. The amino acids altered by the 9

HDR mutations are highlighted in black, and the 7 mutations (E263V, C264R,

GA268/269QT, P273T, R276Q, D278G and D278Y) of ZnF1 generated for additional

functional studies (Figures 4 and 5) are highlighted in grey.


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Figure 2

Detection of GATA3 mutation in exon 3 in family 8/2000 with HDR by restriction enzyme

analysis. DNA sequence analysis of individual III.1 revealed a G to T transversion at codon

228, thus altering the wild-type (WT) sequence GAG, encoding a glutamine to the mutant

(m) sequence TAG, which is a termination (Stop) codon. This nonsense mutation also

resulted in the loss of the wild-type BsoBI restriction enzyme (C/CCGAG) and this

facilitated the confirmation of the mutation (b). PCR amplification and BsoBI digestion

would result in two products of 167bp and 100bp from the normal i.e. wild type (WT)

sequence, but an additional band of 267bp would be expected from the mutant (m) sequence

as is illustrated in the restriction map in (c). Cosegregation of this E228X mutation and its

heterozygosity in the affected members (II.2 and III.1) was demonstrated (b), and the

absence of this E228X mutation in 110 alleles from 55 unrelated normal individuals (N1 and

N2 shown) indicates that it is not a common DNA sequence polymorphism. Similar

restriction enzyme analysis was used to confirm and demonstrate cosegregation of the

codon 201 deletion, and the C318R and the R367X mutations (Table II). Individuals are

represented as: male (squares), female (circles), unaffected (open symbols), affected with

hypoparathyroidism (filled upper-left quadrant), affected with deafness (filled lower-left

quadrant), affected with renal anomalies (filled lower-right quadrant), deceased (slash

through symbol) and not available (NA).


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Figure 3

Detection of acceptor splice site mutation at the intron 5/exon 6 boundary in family 16/2001

(Table II). DNA sequence analysis of the affected proband (Table I) revealed a g to t

transversion at the –1 position, which resulted in an alteration of the invariant ag acceptor

splice site (a). Analysis of 110 alleles from 55 unrelated normals revealed the presence of

the normal ag acceptor splice site and an absence of the at sequence, thereby indicating that

the g to t transversion at position –1 was not a common sequence polymorphism but a likely

mutation that would alter mRNA splicing (data not shown). In addition, an examination of

the DNA sequences of codons 351 to 353 revealed another naturally occurring, but

normally unused, acceptor splice site sequence (ncag)(61,62). The effects of the likely

mutation were therefore investigated by using wild-type (WT) and mutant (m) minigene

constructs containing exons 4, 5, and 6 in the mammalian expression vector pcDNA 3.1,

and transfecting these into COS-1 cells. Total RNA was extracted from the cells and

utilised with exon 5 and exon 6 specific primers in RT-PCRs. The mutant RT-PCR

products are smaller and DNA sequence analysis of these revealed splicing of exon 5 to an

internal site in exon 6 that resulted in a new sequence which encoded a missense peptide

with a premature termination at codon 367. Thus, the mutation had resulted in utilisation of

an alternative, naturally occurring, but normally non-utilised, acceptor splice sequence.

Exon sequence (uppercase), intron sequence (lower case); + with reverse transcriptase; -

without reverse transcriptase; size markers are in bp.


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Figure 4

Analysis of DNA-binding properties and subcellular localisation of GATA3 mutants

associated with HDR. Western blot analysis (a) of in vitro translated wild-type (WT) and

GATA3 mutants revealed the expected 50kDa WT product. The missense mutations

C318R and N320K also yield a 50kDa product, whereas the nonsense (E228X and R367X)

and frameshift deletions (201DC and 355DCT) yield the predicted truncated products (Table

II). Electrophoretic mobility shift assays, EMSAs (b). COS-1 cells were transfected with

either the WT or mutant GATA3 constructs and nuclear extracts prepared for binding

reactions which used a radio-labelled (3 2P) double-stranded oligonucleotide containing the

GATA consensus DNA sequence (16). Control binding reactions using untransfected (UT)

cells and the oligonucleotide alone (OA) i.e. without nuclear extract were performed. The

WT GATA3 bound to ds DNA and the method was sensitive enough to detect 10% of the

WT GATA3 binding reaction. GATA3 mutants, which disrupted or lead to a loss of ZnF2

(Figure 1), all resulted in a loss of DNA binding. However, EMSAs revealed normal DNA

binding by all the GATA3 ZnF1 missense mutants, whether they were associated with HDR

(W275R) or had been engineered (E263V, C264R, GA268/269QT, P273T, R276Q, D278G

and D278Y), data shown for C264R and W275R (panel c, 0 mins). The stability of the

DNA binding of all these 8 GATA3 mutants that occur in ZnF1 were further studied using

dissociation gel shift assays (c) in which unlabelled dsDNA was added, and the effects on

the binding of GATA3 to the radiolabelled dsDNA measured over a time course of 60

minutes by autoradiography. The wild-type (WT) GATA3 and mutants GA268/269QT,

W275R, D278G and D278Y dissociated from the radio-labelled DNA at similar rates,

whereas the E263V, C264R, P273T and R276Q mutations, dissociated more rapidly such

that the 100-fold excess of unlabelled DNA had replaced all, or a substantial amount, of the

radio-labelled DNA by 30 minutes. Subcellular localisation studies (d) revealed that WT-

GATA3 and the 12 mutants (E263V, C264R, GA268/269QT, P273T, W275R, R276Q,

D278G, D278Y, C318R, N320K, 355DCT, and R367X) that contained ZnF1 (Figure1)


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30

accumulated in the nucleus, whilst the 2 mutants (D201C and E228X) that lacked ZnF1 did

not accumulate in the nucleus but instead had a pattern similar to that observed in the cells

transfected with GFP alone (GFP). Green and blue labelling represent GFP and nuclear

DAPI staining, respectively. Nuclear GFP staining masks DAPI staining, and hence the

presence of blue nuclei represents untransfected cells. The scale bar represents 10µm.


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Figure 5

Interactions between GATA3 ZnF1 and FOG2 ZnFs using a yeast two-hybrid assay. The

interaction between wild-type (WT) or mutant GATA3 N-terminal ZnF1, and FOG2 ZnFs

1, 5, 6, and 8 was studied in the yeast reporter strain AH109 following transformation with

the vectors containing GATA3 ZnF1 (pGBKT7) and each FOG2 ZnF (pGADT7) in turn.

Yeast growth was monitored 48 hours after streaking and incubation at 30˚C using either

double dropout, DDO (Leu-Trp-), media (a) as a control, or quaternary drop out, QDO (Leu-

Trp-Ade-His-) media (b) in which growth is dependent on the physical interaction between

the GATA3-Gal4 DNA-BD and FOG2-Gal4-AD fusion proteins (29,63). The SV40 large

T antigen and p53 proteins which are known to interact (32) were used as positive controls.

Co-expression of the GATA3 and FOG2 Gal4 fusion proteins in the yeast colonies was

confirmed in each case by Western blot analysis. The WT GATA3 fusion protein interacted

with FOG2 ZnF 1, 5, 6, and 8 fusion proteins, whereas the engineered mutant E263V,

C264R and GA268/269QT GATA3 proteins showed an absence of interaction with these

FOG2 ZnFs. However, the W275R mutant, which was reported in an HDR patient (22),

significantly interacted with FOG2 ZnF6 but not with FOG2 ZnFs 1, 5, and 8. The

engineered mutants P273T and R276Q interacted with FOG2 ZnFs 1, 5, 6, and 8, whilst

D278G and D278Y interacted with FOG2 ZnFs 1, 5, and 6. These results were confirmed

by GST pull-down assays (c) that utilised in vitro translated 3 5S-labelled GATA3 and GST-

FOG2 ZnF fusion proteins (data shown for wild-type, C264R and W275R). The input row

demonstrates that equal amounts of the wild-type and mutant GATA3 protein were loaded,

and the Coomassie-stained gel (d) shows that approximately equal amounts of GST-FOG2

fusion proteins were used in the GST pull-down assay. These results of GATA3-ZnF1

interactions with FOG2, are consistent with the findings of the proposed GATA1-ZnF1 3-

dimensional model (Figure 6).


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Figure 6

Three-dimensional structure of the human GATA3-ZnF1 based on the model of murine

GATA1 ZnF1 (37). Human GATA1, which consists of 413 amino acids, and human

GATA3, which consists 444 amino acids belong to the same sub-family (10) and share

structural similarities which include two ZnFs (Figure 1). The 3-dimensional structure of

the murine GATA1-ZnF1(residues 201 to 243) has been characterised and this has 91%

identity to the human GATA3-ZnF1 (a), thereby enabling us to use this to construct a 3-

dimensional model of human GATA3-ZnF1 (residues 261-303). The residues shown in

the ribbon (b) and space-filing (c) models refer to those of the equivalent human (h)GATA3

ZnF1, and the corresponding murine (m)GATA1 ZnF1 residues are as follows

hE263=mE203, hC264=mC204, hG268=mG208, hA269=mA209, hW275=mW215,

hR276=mR216, hD278=mD218. Residues participating in the interaction between

mGATA1 and FOG ZnFs, which include the human equivalents of E263, C264, G268, and

A269 are shown. They are seen to form a binding surface distinct from that containing

W275 and D278 which have been shown to interact with different FOG zinc fingers, whilst

R276 lies at the DNA-binding surface and does not participate in binding to FOG2 ZnFs.

P273, which also resides at a DNA binding surface is not visible in the projection shown.

The backbone is shown as dark magenta; hydrophobic side chains as grey; polar side

chains as magenta; acidic side chains as red; and basic side chains as blue. This color

scheme derives from the CPK color scheme as follows. hydrophobic = carbon; acidic =

oxygen; basic = nitrogen. polar but uncharged = a mixture of oxygen (red) and nitrogen

(blue), namely magenta. The backbone is polar but less likely (dark magenta) than side

chains to hydrogen bond to non-backbone moieties, as most backbone hydrogen bonding

occurs within the backbone.


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Table IClinical and Biochemical findings in 10 HDR patients

Family/Patient(sex)

Hypoparathyroidism Deafness (Sensorineural) Renal abnormalitiesA

Serum Ca2+

(mmol/l)Serum PTHB

(Assay range)PresentationC AgeD SymmetryE AgeD C, H, A, R, S, ERF,

(AgeD)1 8/2000

Proband (F) 1.01 13pg/ml (10-64) Se 20 B 4 H+ERFMother (F) 1.97 28pg/ml (10-64) As 45 B <25 None

2 13/2001 (M) 1.05 6pg/ml (10-64) Se/Te 13 B, R>L <1 None

3 2/2001Proband (F) 1.42 1.1pmol/l (1.0-7.0) As 3 B 3 S+R+C (3)Father (M) 2.00 1.1pmol/l (1.0-7.0) As <40 B, L>R <30 R

4 16/1998 (F) 1.25 UD Se 4 B <8 A

5 19/1992 (M) 1.91 UD As 11 B 8 S+R+H (0.2), +ERF (9)

6 19/2000Proband (F) 1.60 8pg/ml (10-64) As 27 B 4 C (23)Mother (F) 1.97 15pg/ml (10-64) As 51 B 24 None

7 16/2001 (F) 1.72 13pg/ml (10-64) Se 1 B, R>L 5 A

Normal range 2.15-2.65

A Renal abnormalities: C-cysts, H-hypoplasia, A-agenesis, R-vesicoureteric reflux, S-sepsis, ERF-end stage renal failure. B PTH normal range given inparenthesis, UD-undetectable. C As-asymptomatic, Se-seizures, Te-tetany. D Age (years) at diagnosis. E B-bilateral, R-right, L-left.


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Table IIGATA3 abnormalities detected in HDR patients

MutationnumberA

Family/PatientB Exon Codon Base Change Amino-acidchange

RE/ASO/ARMS/SAC

Predicted effect

Nonsense mutation1 8/2000 3 228 GAG‡TAG Glu‡Stop RE E228X; Loss of ZnF1 and ZnF2; HI2 13/2001D 6 367 CGA‡TGA Arg‡Stop RE R367X; Loss of basic amino acids flanking

ZnF2, HI

Intragenic deletions (D)3 2/2001 3 201 TCC‡T-C Frameshift RE Missense peptide 3 amino acids from 202-204,

followed by premature stop at codon 205.Truncated protein with loss of ZnF1 andZnF2; HI

4 16/1998 6 355 CTG‡--G Frameshift ARMS Missense peptide 15 amino acids from 355-369,followed by premature stop at codon 370.Truncated protein with loss of C-terminal basicamino acids; HI

Missense mutation5 19/1992 5 318 TGT‡CGT Cys‡Arg RE C318R; Loss of Zn2+ coordination; Disruption

of ZnF2; HI6 19/2000 5 320 AAC‡AAA Asn‡Lys ASO N320K; Disruption of ZnF2; HI

Splice site acceptor mutation7 16/2001D Intron 5

/Exon 6Boundary

ag‡at Frameshift fromcodon 351

SA Missense peptide 18 amino acids, followed bypremature stop at codon 367. Loss of basicamino acids flanking ZnF2; HI

A Mutation number refers to location shown in Figure 1. B Family identification refers to clinical details shown in Table I. C Analysis by restriction enzymes(RE), or allele specific oligonucleotide (ASO) hybridisation, or amplification refractory mutation system (ARMS)-PCR analysis, or sequence analysis (SA).D de novo mutation not present in either parent of proband. Haploinsufficiency (HI).


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E G R E CV

NC

GATS

TP L W R R

GT

A

H

T S

Q

W R

DPV

G

YL

CN

C G L Y A G CA

NC

TTT

TT L R N A

NG

K I M I QAN

C

C

G L Y Y LH H N N R P L T K E G T R N R MK SS K S K K

T P D Y E E G R H P D Q K P K N V X

D

.. . . . . . . . . ..fs fs

350 360

K3707 4 2,b

340

330

290

280

270

260

f a

6Zn++ Zn++

5

ZnF1 ZnF2Figure 1

Nonsense = 4del, fs = 3ins, fs = 1

del, inf = 1Missense = 3

Acceptor splice site = 1del, whole gene = 6

Total = 19

Exon 1 2 3 4 5 6ATG TAG

TA1 TA2 ZnF1 ZnF2

1 a 2,bc 3 4

d

fe5 6

7

e


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G to T transversion (E228X)

BsoBI100bp 167bp267bp

WT

WT

m

WTm

TAC GTG CCC AGG

T

Tyr Val ProGlu

Stop

225 226 227 228

Wild type (WT)

Codon

Mutant (m)

BsoBI

Amino acid(WT)

(m)

Family 8/2000

267bp

167bp

100bp

N1 N2

I

III

II

21

1 2

NA

a

1 2

b

c

Figure 2


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Exon 5 Intron 5 Exon 6Codon

+ - + -m WT

300

800700600

500

400

Unspliced

Spliced

... CTT CAC AAT gt...

...ag ATT AAC AGA CCC CTG ... CGA

...at att aac ag ACC CCT ... TAGmutant (m)

wild type (WT)

...Leu His Asn

Thr Pro ... Stop

...348 349 350

Ile Asn Arg Pro Leu ... Arg

351 352 353 354 355 ... 367

S

Acceptor splice site mutationFigure 3

-

--

-

-

---

- - - -- - - -

- - - -- - - -

- - - - -- - - - -

- - - - - -- - - - - -

- - - - - -- - - - - -


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WT 201∆C

E228X

C318R

N320K

355∆CT

R367X

WT UT 10%

48.5

33.4

20.8

Bound ds DNA+ GATA3

Unbound ds DNA

0 10 30 60 0 10 30 60 0 10 30 60 min

0 10 30 60 0 10 30 60 0 10 30 60 min

0 10 30 60 0 10 30 60 0 10 30 60 min

WT E263V C264R

GA268/269QT P273T W275R

R276Q D278G D278Y

a

b

c d

Figure 4

OA


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pGBKT7 Lam p53 GATA3ZnF WT

E263V C264R GA268/269QT

P273T W275R R276Q D278G D278Y

pGBKT7 Lam p53 GATA3ZnF WT

E263V C264R GA268/269QT

P273T W275R R276Q D278G D278Y

pGADT7

Large T

FOG2 ZnF1

FOG2 ZnF5

FOG2 ZnF6

FOG2 ZnF8

pGADT7

Large T

FOG2 ZnF1

FOG2 ZnF5

FOG2 ZnF6

FOG2 ZnF8

DDO

QDO

GATA3 ZnF1 mutants

GATA3 ZnF1 mutants

WT C264R W275R

GATA3

Input

1

5

6

8

GST

FOG2ZnF

1 5 6 8 GSTFOG2 ZnF

a

b

c d

Figure 5 by guest on M

arch 23, 2018http://w

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R276

R276

W275W275

D278D278

C264 C264E263 E263

A269G268

b c

hGATA3 ZnF1 GRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNGQNRPLIK-RECVNCGAT-TPLWRRD-TGHYLCNACGLYHKMNGQNRPLI-

mGATA1 ZnF1 ARECVNCGATATPLWRRDRTGHYLCNACGLYHKMNGQNRPLIR

aFigure 6


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McWillliams, Susan Rigden, Julian Sampson, Andrew Williams and Rajesh V. ThakkerCrowe, Angus Dobbie, Geeta Hampson, Ian Holdaway, Michael A. Levine, Robert

M. Andrew Nesbit, Michael R. Bowl, Brian Harding, Asif Ali, Alejandro Ayala, Caroldysplasia (HDR) syndrome

Characterisation of GATA3 mutations in the hypoparathyroidism, deafness and renal

published online February 24, 2004J. Biol. Chem.

10.1074/jbc.M401797200Access the most updated version of this article at doi:

Alerts:

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