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.

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
Polarized hyphal growth
Histone H1-GFP tagged nuclei
Chromatin is heterogeneous
euchromatin
decondensed, active,
DNA unmethylated
histones hyperacetylated
heterochromatin
condensed, inactive,
DNA methylated
histones hypoacetylated
Arabidopsis
Wolffe (1998) Chromatin
fission yeast
mouse
Nuclear architecture
Chromocenter
Region of
heterochromatin
that may be
associated with
the telomeres
HP1-GFP
Histone H1-GFP
Role of heterochromatin
in polarized growth
HP1 mutants exhibit slow linear growth
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
1. PCR
2. Digest plasmid and insert
5’
3’
3. Ligate
XbaI BamHI
3. Transformation into
competent E.coli
pMF272
pGF1
DH5
DH5
DH5
6. Analysis by epifluorescent microscopy
4. Purify DNA, linearize
5. Transform N. crassa
Amplification of genes for
centromere-specific proteins
M
+ all three genes were amplified
successfully
+ Cenp-S was fused to GFP
+ Cenp-I was fused to RFP
+ CAC-3 fusions did not work
3.0 kb
2.0 kb
1.0 kb
0.5 kb
CCG1 promoter RFP
Cenp-I
CCG1 promoter Cenp-S GFP
C-Terminus
C-Terminus
CENPS
CENP-I
CAC-3
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
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
Cenp-S-GFP
X
X
N2557 (rid his-3 mat a)
N2556 (his-3 mat a; hpoRIP2)
RFP-Cenp-I
RFP-Cenp-I
X
X
N2557 (rid his-3 mat a)
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.
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 of
Cenp-S
Imaging of the Labeled
Centromeres
Cenp-I localized in
the centromeric
DNA region.
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.
Cenp-I and HP1 co-localize partially
HP1-GFP
RFP-CENP-I
SON-1-GFP + RFP-CENP-I
SON-1 gfp

CENP-I rfp
Cenp-I localization is centromeric
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
CENP-S-GFP; hpo
RFP-CENP-I; hpo

The same appears to be true for CenH3 (data not shown)

Centromere localization appears independent of heterochromatin.
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

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

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




Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Michael Freitag
Thomas Lew