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Heterochromatin distribution and function in interphase. Grant Farr (Freitag Lab). Neurospora crassa image: N. B. Raju, Stanford University. Long-term objectives. To find fungus-specific inhibitors to better combat fungal infections in humans, animals and plants.

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heterochromatin distribution and function in interphase

Heterochromatin distribution and function ininterphase

Grant Farr

(Freitag Lab)

Neurospora crassa image:

N. B. Raju, Stanford University

long term objectives
Long-term objectives
  • To find fungus-specific inhibitors to better combat fungal infections in humans, animals and plants.
  • To determine if centromere and heterochromatin organization are involved in polarized growth.

Aspergillosis

X-Ray by ADAMS Health Care Center

neurospora crassa

Vegetative cycle

Histone H1-GFP tagged nuclei

M. Springer

Neurospora crassa
slide4

Polarized hyphal growth

Histone H1-GFP tagged nuclei

chromatin is heterogeneous
Chromatin is heterogeneous

euchromatindecondensed, active, DNA unmethylatedhistones hyperacetylated

heterochromatincondensed, inactive, DNA methylatedhistones hypoacetylated

Arabidopsis

fission yeast

mouse

Wolffe (1998) Chromatin

nuclear architecture
Nuclear architecture

Chromocenter

Region of heterochromatin that may be associated with the telomeres

HP1-GFP

Histone H1-GFP

role of heterochromatin in polarized growth
Role of heterochromatin in polarized growth

HP1 mutants exhibit slow linear growth

centromere specific proteins
Centromere-specific proteins

CENP-A (CenH3)

Centromere-specific histone H3

Other proteins:

~30 proteins (CENP-B to CENP-S) in humans

~60 known proteins in

yeast

Neurospora shares four identifiable proteins with humans:

CenH3

Cenp-I

Cenp-S

CAC-3 (CAFp46/48)

Young-Tae Chang and Young-Soo Kim from NYU department of Chemistry

experimental outline

DH5

DH5

DH5

Experimental outline

1. PCR

2. Digest plasmid and insert

3. Ligate

5’

3’

XbaI

BamHI

3. Transformation into competent E.coli

pMF272

pGF1

6. Analysis by epifluorescent microscopy

4. Purify DNA, linearize

5. Transform N. crassa

amplification of genes for centromere specific proteins
Amplification of genes for centromere-specific proteins

+ all three genes were amplified successfully

+ Cenp-S was fused to GFP

+ Cenp-I was fused to RFP

+ CAC-3 fusions did not work

M

CENP-S

CENP-I

CAC-3

3.0 kb

2.0 kb

1.0 kb

0.5 kb

CCG1 promoter

RFP

Cenp-I

C-Terminus

CCG1 promoter

Cenp-S

GFP

C-Terminus

transformation of neurospora crassa
Transformation of Neurospora crassa
  • Transformed linearized plasmid DNA carrying Cenp-S and Cenp-I fusion genes into two N. crassa strains each
  • Genes targeted to the

his-3 locus

Initial transformations of Cenp-S

Initial transformation of CENP-I

backcross to purify strains
Backcross to purify strains

Uncrossed CENP-I RFP

Crossed CENP-I with expected localization and less background noise

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

  • Crosses:

Cenp-S-GFP X N2557 (ridhis-3 mat a)

Cenp-S-GFP X N2556 (his-3 mat a; hpoRIP2)

RFP-Cenp-I X N2557 (ridhis-3 mat a)

RFP-Cenp-I X N2556 (his-3 mat a; hpoRIP2)

cenp s labels the centromere
The HP1 cells previously characterized have similar localization.

CenH3 localization is identical to Cenp-S localization.

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

Cenp-S labels the centromere

HP1 labeled with centromere

and telomeric regions fluorescing

Cenp-S-GFP

imaging of the labeled centromeres
Imaging of the Labeled Centromeres

Cenp-S gfp localized at the centromere

imaging of the labeled centromeres16
Imaging of the Labeled Centromeres

Cenp-I localized in the centromeric DNA region.

confirming centromeric localization
Confirming centromeric localization

Patrick Hickey (University of Edinburgh)

  • Hypha of different strains can form heterokaryons.
  • Fusing SON-1-GFP and HP1-GFP strains with the new Cenp-S and Cenp-I strains will show simultaneous localization of the nuclear membrane and centromeres.
son 1 gfp rfp cenp i
SON-1-GFP + RFP-CENP-I
  • Cenp-I localization is centromeric

SON-1 gfp

CENP-I rfp

is hp1 required for centromere localization
Is HP1 required for centromere localization?
  • Hypothesis:

Heterochromatin is required for centromere localization.

  • I tested this with the hpo X Cenp-S and Cenp-I crosses.
localization of cenp s and cenp i is maintained in hp1 mutants
Localization of CENP-S and CENP-I is maintained in HP1 mutants
  • The same appears to be true for CenH3 (data not shown)
  • Centromere localization appears independent of heterochromatin.

CENP-S-GFP; hpo

RFP-CENP-I; hpo

possible cenp i mutant
Possible Cenp-I mutant
  • One of the Cenp-I X wildtype crosses gave us an hpo mutant like growth phenotype.
  • The phenotypes are separable by microscopy.
  • In the future we will sequence the Cenp-I genes to search for point mutations introduced by RIP in the cross.
summary
Summary
  • The Cenp-I and Cenp-S fusion proteins were expressed and appear to be located at centromeres.
  • Lack of heterochromatin binding protein does not affect the localization of either proteins.
  • Possible mutant of Cenp-I affects apical growth.
future studies
Future Studies
  • Construct an RFP-Cenp-S strain and a Cenp-I-GFP strains to better understand localization of the two using heterokaryons.
  • Slow-growing strains will be tested for the hpo mutation by DNA sequencing to verify that Cenp-S and Cenp-I localization were independent of HP1.
  • Use protein affinity tags (TAP, Myc, HA) to characterize other proteins in centromere complexes.
  • Find mutants in the Neurospora “knockout” collection that affect centromere localization and polarized growth.
acknowledgements
Acknowledgements
  • Howard Hughes Medical Institute
  • Dr. Kevin Ahern
  • Dr. Michael Freitag
  • Thomas Lew