Heterochromatin distribution and function in interphase Grant Farr (Freitag Lab) Neurospora crassa image: N. B. Raju, Stanford University.

Heterochromatin distribution and

function in interphase

Grant Farr (Freitag Lab)

Neurospora crassa image:

N. B. Raju, Stanford University

Long-term objectivesLong-term objectives

To find fungus-specific To find fungus-specific inhibitors to better inhibitors to better combat fungal combat fungal infections in humans, infections in humans, animals and plants.animals and plants.

To determine if To determine if centromere and centromere and heterochromatin heterochromatin organization are organization are involved in polarized involved in polarized growth.growth.

X-Ray by ADAMS Health Care Center

Aspergillosis

Histone H1-GFP tagged nuclei

Vegetative cycle

M. Springer

Neurospora crassa

Polarized hyphal growth

Histone H1-GFP tagged nuclei

Chromatin is Chromatin is heterogeneousheterogeneous

Wolffe (1998) Chromatin

euchromatindecondensed, active, DNA unmethylatedhistones hyperacetylated

heterochromatincondensed, inactive, DNA methylatedhistones hypoacetylated

Arabidopsis fission yeast mouse

Nuclear architectureNuclear architecture

HP1-GFP

Histone H1-GFP

Region of heterochromatin that may be associated with the telomeres

Chromocenter

Role of heterochromatin Role of heterochromatin in polarized growthin polarized growth

HP1 mutants exhibit slow linear growth

Centromere-specific Centromere-specific proteinsproteins

CENP-A (CenH3)CENP-A (CenH3)Centromere-specific histone Centromere-specific histone H3H3

Other proteins:Other proteins:~30 proteins (CENP-B to ~30 proteins (CENP-B to

CENP-S) in humansCENP-S) in humans~60 known proteins in~60 known proteins in

yeastyeast

NeurosporaNeurospora shares four shares four identifiable proteins with identifiable proteins with humans:humans:CenH3CenH3Cenp-ICenp-ICenp-SCenp-SCAC-3 (CAFp46/48)CAC-3 (CAFp46/48) Young-Tae Chang and Young-Soo

Kim from NYU department of Chemistry

Experimental outlineExperimental outline

5’ 3’

2. Digest plasmid and insert

pGF1

XbaI

BamHI

3. Ligate

pMF272

3. Transformation into competent E.coli

DH5

DH5

DH5

4. Purify DNA, linearize

5. Transform N. crassa

6. Analysis by epifluorescent microscopy

1. PCR

Amplification of genes Amplification of genes for centromere-specific for centromere-specific

proteinsproteins

++ all three genes were all three genes were amplified successfullyamplified successfully

+ Cenp-S was fused to GFP+ Cenp-S was fused to GFP

+ Cenp-I was fused to RFP+ Cenp-I was fused to RFP

+ CAC-3 fusions did not work+ CAC-3 fusions did not work

CENP-S

CENP-I CAC-3M

0.5 kb

1.0 kb

2.0 kb

3.0 kb

Cenp-ICCG1 promoter RFP C-Terminus

GFPCCG1 promoter Cenp-S C-Terminus

Transformation of Transformation of Neurospora crassaNeurospora crassa

Transformed Transformed linearized plasmid linearized plasmid DNA carrying Cenp-S DNA carrying Cenp-S and Cenp-I fusion and Cenp-I fusion genes into two genes into two N. N. crassacrassa strains each strains each

Genes targeted to theGenes targeted to the his-3his-3 locus locus

Initial transformations of Cenp-S

Initial transformation of CENP-I

Backcross to purify Backcross to purify strainsstrains

Crosses:Crosses:Cenp-S-GFP Cenp-S-GFP XX N2557 (N2557 (ridrid his-3 mat ahis-3 mat a))Cenp-S-GFPCenp-S-GFP XX N2556 (N2556 (his-3 mat ahis-3 mat a; ; hpohpoRIP2RIP2))

RFP-Cenp-IRFP-Cenp-I XX N2557 (N2557 (ridrid his-3 mat ahis-3 mat a))RFP-Cenp-IRFP-Cenp-I XX N2556 (N2556 (his-3 mat ahis-3 mat a; ; hpohpoRIP2RIP2))

A.J.F. Griffiths, U.B.C.

Uncrossed CENP-I RFP

Crossed CENP-I with expected localization and less background noise

Cenp-S labels the Cenp-S labels the centromerecentromere

The HP1 cells The HP1 cells previously previously characterized have characterized have similar localization. similar localization.

CenH3 localization is CenH3 localization is identical to Cenp-S identical to Cenp-S localization.localization.

Cenp-S is identified as a Cenp-S is identified as a part of the centromeric part of the centromeric Cenp-A complex.Cenp-A complex.

HP1 labeled with centromere and telomeric regions fluorescing

Cenp-S-GFP

Imaging of the Labeled Centromeres

Cenp-S gfp localized at the centromere

Centromeric localization Centromeric localization of Cenp-Sof Cenp-S

Imaging of the Labeled Imaging of the Labeled CentromeresCentromeres

Cenp-I localized in the centromeric DNA region.

Confirming centromeric Confirming centromeric localizationlocalization

Hypha of different strains can form heterokaryons.Hypha of different strains can form heterokaryons.

Fusing SON-1-GFP and HP1-GFP strains with the Fusing SON-1-GFP and HP1-GFP strains with the new Cenp-S and Cenp-I strains will show new Cenp-S and Cenp-I strains will show simultaneous localization of the nuclear simultaneous localization of the nuclear membrane and centromeres. membrane and centromeres.

Patrick Hickey (University of Edinburgh)

Cenp-I and HP1 co-localize Cenp-I and HP1 co-localize partially partially

RFP-CENP-I

HP1-GFP

SON-1-GFP + RFP-CENP-SON-1-GFP + RFP-CENP-II

Cenp-I localization is centromericCenp-I localization is centromeric

SON-1 gfp CENP-I rfp

Is HP1 required for Is HP1 required for centromere localization?centromere localization?

Hypothesis: Hypothesis:

Heterochromatin is required for Heterochromatin is required for centromere localization. centromere localization.

I tested this with the I tested this with the hpohpo X Cenp-S X Cenp-S and Cenp-I crosses. and Cenp-I crosses.

Localization of CENP-S and Localization of CENP-S and CENP-I CENP-I

is maintained in HP1 mutantsis maintained in HP1 mutants

The same appears to be true for CenH3 (data not shown)The same appears to be true for CenH3 (data not shown)

Centromere localization appears independent of Centromere localization appears independent of heterochromatin.heterochromatin.

RFP-CENP-I; hpo CENP-S-GFP; hpo

Possible Cenp-I mutantPossible Cenp-I mutant One of the Cenp-I X One of the Cenp-I X

wildtype crosses gave wildtype crosses gave us an us an hpohpo mutant like mutant like growth phenotype.growth phenotype.

The phenotypes are The phenotypes are separable by separable by microscopy.microscopy.

In the future we will In the future we will sequence the Cenp-I sequence the Cenp-I genes to search for genes to search for point mutations point mutations introduced by RIP in introduced by RIP in the cross.the cross.

SummarySummary The Cenp-I and Cenp-S fusion proteins The Cenp-I and Cenp-S fusion proteins

were expressed and appear to be located at were expressed and appear to be located at centromeres.centromeres.

Lack of heterochromatin binding protein Lack of heterochromatin binding protein does not affect the localization of either does not affect the localization of either proteins.proteins.

Possible mutant of Cenp-I affects apical Possible mutant of Cenp-I affects apical growth.growth.

Future StudiesFuture Studies Construct an RFP-Cenp-S strain and a Cenp-I-GFP Construct an RFP-Cenp-S strain and a Cenp-I-GFP

strains to better understand localization of the strains to better understand localization of the two using heterokaryons.two using heterokaryons.

Slow-growing strains will be tested for the Slow-growing strains will be tested for the hpohpo mutation by DNA sequencing to verify that Cenp-S mutation by DNA sequencing to verify that Cenp-S and Cenp-I localization were independent of HP1.and Cenp-I localization were independent of HP1.

Use protein affinity tags (TAP, Myc, HA) to Use protein affinity tags (TAP, Myc, HA) to characterize other proteins in centromere characterize other proteins in centromere complexes.complexes.

Find mutants in the Find mutants in the Neurospora “Neurospora “knockout” knockout” collection that affect centromere localization and collection that affect centromere localization and polarized growth.polarized growth.

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Kevin AhernDr. Kevin Ahern Dr. Michael FreitagDr. Michael Freitag Thomas LewThomas Lew