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EPIGENETIC GENE SILENCING
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Rohinton
Kamakaka, PhD, Head, Unit on
Chromatin and Transcription Namrita
Dhillon, PhD, Research Fellow Sunil
Gangadharan, PhD, Visiting Fellow Devyani
Haldar, PhD, Visiting Fellow Masaya
Oki, PhD, Visiting Fellow Naoe Kotomura, PhD, Guest Researcher |
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Our
laboratory is devoted to understanding the mechanisms by which entire regions
of the genome are rendered inaccessible to transcription and recombination.
Silencing of genomic domains requires a complex series of interactions
between inactivation centers called silencers and numerous repressor
proteins. The silencers recruit repressor protein complexes composed of the
Sir proteins that interact with histones in nucleosomes to form a chromatin
domain that is both inaccessible and inert to various cellular processes. In
an effort to understand in molecular detail the mechanism by which silencing
is effected, we are currently focusing on the Sir proteins and their
interactions with the histones. Histone
variants and cell cycle progression Dhillon Numerous
histone variants differ in amount and localization; moreover, their
expression is regulated, suggesting a role in gene regulation. We isolated
the histone variant Htz1p as a suppressor of a Sir1p mutant, and
recent results suggest that the protein is localized to regions flanking the
silenced loci. We are currently interested in understanding the many roles of
Htzlp in the cell. Cells lacking the protein are hypersensitive to various
drugs such as hydroxyurea (HU) and benomyl. We have generated point mutants
in the protein that are sensitive to these drugs; we find that they all map
to a single domain and are suppressed by high doses of genes that are
regulated in response to DNA damage and environmental stress. While
cells lacking Htz1 are unable to grow on media containing sublethal amounts
of HU, the protein is not involved in mediating the checkpoint response that
is normally triggered when cells are exposed to higher doses of the drug. All
the HU-mediated effects, such as the S-phase arrest, Rad53 phosphorylation,
and firing of early but not late origins observed in wild-type cells, are
also seen in htz1 mutants. However, we have uncovered genetic
interactions between Htz1 and proteins involved in replication checkpoint
control, and our analyses suggest that cells lacking the protein have
increased defects during S-phase. Cells
lacking Htz1 progress more slowly through S-phase, and the timing of
replication of both early- and late-replicating loci is delayed in the
mutants. While Htz1 is present at both early and late origins, the delay in
replication in htz1 mutants is not attributable to impaired ORC
binding to origins, as indicated by ChIP experiments. Strikingly, however,
the release of Cdc45 from both early and late origins is slower in htz1
cells. Our results suggest (1) that the histone variant is required for an
open chromatin state that facilitates origin firing and S-phase progression
and (2) that, in the absence of Htz1, S-phase is slowed. We are currently
attempting to ascertain the nature of the cell cycle defect to gain a better
understanding of the function of this protein in the cell. Characterization
of Sir protein complexes involved in silencing Gangadharan, Kotomura We
are addressing the question as to how Sir proteins function to form silenced
domains. Genetic studies have revealed that distinct combinations of the Sir protein
complexes repress multiple loci. Silencing at all the loci requires Sir2p,
which possesses histone deacetylase activity. In addition, Sir2p is the only
Sir protein to have additional homologs in yeast (Hst1-4p) and the only Sir
protein conserved throughout evolution. We are reconstituting
Sir2p-containing protein complexes to characterize the function of each
individual component within the complexes. Using
the above complexes and histones in nucleosomes, we have begun studies on the
reconstitution of silenced chromatin (Ghidelli et al., EMBO J
2001;20:4522). We are performing in vitro studies of the binding of
purified recombinant Sir proteins to positioned nucleosomes in arrays,
followed by DNaseI foot-printing analysis as well as protein-protein crosslinking
and sedimentation analysis. Our long-term goals call for studies on the
regulation of the Sir enzymes within the cell and in vitro studies
aimed at the eventual development of specific inhibitors of the enzymes that
may be of therapeutic value. Depending on whether it is possible to mimic
exactly the silenced state in vitro, the studies will provide an
important index of our current understanding of transcriptional silencing,
given that mechanisms are rarely established by genetic means and usually
require biochemical tests. Chromatin
domains in silencing Oki, Valenzuela We
are also interested in understanding the mechanism by which silenced
chromatin domains are restricted to specific regions along the DNA fiber.
Eukaryotic chromosomes are organized into discrete domains delimited by
domain boundaries. We have demonstrated that a specific t-RNA gene mediates
barrier functions at the HMR locus (Donze et al., Genes Dev
1999;13:698). The proteins that are required to prevent the spread of
heterochromatin into neighboring euchromatin have also been identified (Donze
and Kamakaka, EMBO J 2001;20:520). Our results suggest that barrier
activity may arise from an underlying competition between chromatin
remodeling and silencing activities at the interface of euchromatin and
heterochromatin. In our ongoing studies on yeast barriers, we have used
genetic screens to isolate other DNA elements from yeast that act as barrier
elements. We
have also performed a systematic genome-wide screen for proteins that could
block the spread of silencing in yeast, an analysis that identified numerous
proteins with efficient silencing blocking activities; some of the proteins
had previously been shown to be involved in chromatin dynamics. We isolated
subunits of Swi/Snf, mediator, and TFIID as well as subunits of the Sas-I,
SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes as barrier proteins. We
demonstrated that histone acetylation as well as chromatin remodeling occurred
at both the synthetic barrier and the native boundaries of the silenced
domains and correlated with a block to the spread of silencing (Oki et al.,
2004). Further
mapping analysis in strains with mutated native barrier elements indicates
that the interface between active and silenced chromatin is a junction of
opposing activities with competition between activities that aid in the
spread of silencing and activities that prevent the spread of silenced
chromatin. Our data suggest that several overlapping mechanisms are involved
in delimiting silenced and active domains in vivo. We
are also analyzing a novel form of gene repression mediated by the dominant
mutant SUM1-1. Sum1p normally functions as a mitotic repressor of
meiotic genes, but SUM1-1 is a neomorphic allele that can repress the MATa1
genes at HMR. SUM1-1 spreads across a large region of DNA; the
repression of MATa1 is not localized but occurs throughout the region.
Our analyses indicate that such repression is specific to the MATa1
gene, given that URA3 or ADE2 is not stably repressed at HMR.
Whether the repression is attributable to promoter-specific repression or an
inability to inherit the repressed state is not yet clear and is under
investigation. Oki M, Valenzuela L, Chiba T, Ito T, Kamakaka RT. Barrier
proteins remodel and modify chromatin to restrict silenced domains. Mol
Cell Biol 2004;24:1956-1967. Analysis
of Sir2p in other eukaryotes Haldar A
complete understanding of transcriptional repression requires analysis of several
unrelated loci in different and distinct systems so that salient principles
of repression can be distinguished from organism- and locus-specific
variation. Silencing of chromatin domains in S. pombe shares many
similarities with heterochromatin formation and position effect variegation
in other eukaryotes such as S. cerevisiae and Drosophila.
Functional homologs of the various S. cerevisiae genes that affect
repression are being identified in S. pombe to allow the
purification and characterization of protein complexes containing Sir
proteins. We will couple the studies with mechanistic investigations on
nucleosomal binding in this organism. Publications
Related to Other Work Dhillon N, Kamakaka RT. Breaking through to the other side:
silencers and barriers. Curr Opin Genet Dev 2002;12:188-192. Donze D, Kamakaka RT. Braking the silence: how heterochromatic
gene repression is stopped in its tracks. Bioessays 2002;24:344-349. Gangadharan S, Ghidelli S, Kamakaka RT. Purification of Sir2
proteins from yeast. Methods Enzymol 2004;377:234-254. Kamakaka RT. Chromatin: a connection between loops and barriers?
Curr Biol 2002;12:R535-R537. Kamakaka RT. Heterochromatin: proteins in flux lead to stable
repression. Curr Biol 2003;13:R317-R319. Oki M, Kamakaka RT. Blockers and barriers to transcription:
competing activities? Curr Opin Cell Biol 2002;14:299-304.
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