Gene Silencing 1

 

Repression: Promoter- and sequencespecific 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.

Link

between 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