 Repression: Promoter- and sequence-

specific
 Silencing: Region specific
Large domains of DNA are inaccessible to DNA
binding proteins
Insertion of RNApol I/II promoters leads to
inactivation of the inserted gene
Less accessible to recombination factors
Persist through mitotic and meiotic cell division
 Transcriptionally silent and inaccessible to
DNA-binding factors
 Crucial for centromeric function
 Stablizes repetitive sequences
 Required for proper sister-chromatid
cohesion and chromosome segregation.
 Crucial role in the regulation of gene
expression during development and
cellular differentiation.
Heterochromatin: maintains a condensed appearance
throughout the cell cycle.
In fission yeast and metazoans, heterochromatin is typically
associated with centromeres and telomeres.
Why heterochromatin?
 Constitutive heterochromatin
• Found in centromeres and telomeres
• Displays the same condensed packaging in all
somatic cell type of an organism
 Facultative heterochromatin
• Refers to a region of chromosomes that appears
densely packaged
• Clonally inherited
• Decision is a cell-specific event
Hypoacetylation
Histone
H3-K9 methylation
DNA methylation
• Cytosine is modified
• DNA methyltransferases
Deacetylation
Methylation of H3-K9
H3-mK9 is recognized by HP1
HP1 is a dual function molecule:
• Amino terminal interacts with H3-mK9
• C-terminal possesses chromo shadow box
enabling it to interact with HMT
Initiation
Maintenance
Propagation
Studied in S.pombe.
Required both for initiation chromatin
silencing and assembly of
heterochromatin.
siRNA has been shown to be required
for methylation of H3K9.
Targeting of appropriate enzymes to
specific sequences on the DNA?
DNA methylation has been correlated
with silencing for almost 20 years
now.
Mediates the formation of
multiprotein repression complex.
CpG islands are methylated.
Key protein: Methyl binding proteins
that recognize the methylated DNA.
A proposed model for long-term silencing
of CpG islands. A: This state represents an
active promoter. The active chromatin
marks H3K4me2 and H3K4me3
(represented by a green oval) are present at
this time. This active conformation is not a
target of DNMTs. B: In the permissive
state genes are not being actively expressed
however they are in a conformation which
poises them for transcription. C: During the
susceptible state H3K4me is removed by
lysine demethylases. The nucleosomes now
contain the silencing chromatin marks
H3K9me3, H3K9me2, and H3K27me3. It is
not yet established if these marks are found
together or independently of one another.
This state does not necessarily undergo
DNA methylation, but is susceptible to
DNA methylation. D: In this state the DNA
has become methylated by DNMTs. This
state is not easily reversible. E: DNA
methylation recruits MBPs, chromatin
remodelers, and HDACS which causes a
compact chromatin structure. This allows
for the long-term silencing of the gene.
Linkbetween DNA replication and
heterochromatin state.
ORC proteins
• Mutants in Orc5 have been shown to be
specifically defective either in replication or
in silencing
• The two types of mutants can complement
each other.
Mating type switch
rDNA silencing
Telomere silencing
3 kb heterochromatin region is created
4 SIR proteins
Deletion of SIR2, 3, and 4 abolishes
silencing.
SIR1 has milder phenotype.
None of them binds to DNA.
Sir3p and 4p interact with each other
and with histone octamer.
Sir2p has deacetylase activity.
 Conserved from bacteria to humans
 NAD+ -dependent deacetylases
 Couples deacetylation to hydrolysis of
NAD+.
 Specifically targets H3K9 and K14 as well
as H4 K16.
 In vitro, Sir2 protein possesses ADP
ribosylation activity.
• Relevance in vivo?
 Sir3 and Sir4 have no enzymatic activity.
 Play a structural role in silenced chromatin.
 Sir4:
• Forms complex with Sir2p
• Facilitates association of silencing proteins with
the chromatin.
 Sir3:
• Recruited independently of Sir2p-Sir4p complex
• Promiscuous binding properties