RNAi and chromatin silencing

Heterochromatin - condensed regions during cell cycle (E. Heitz,1928)

- transcriptionally repressed and highly condensed structure

- recombination suppression, chromosome segregation, gene silencing,

- DNA methylation and histone modifications control diverse chromatin states and architectures.

Recent work indicates that this highly condensed chromosomal material is transcribed, and rapidly silenced, by an orchestrated

sequence of events directed by RNA interference (RNAi).

transcription and noncoding RNAs

convergent view of the molecular mechanisms that underlie heterochromatin assembly

- centromeric heterochromatin formation in fission yeast

- RNA-directed DNA methylation in plants - X chromosome inactivation (XCI) and imprinting in mammals.

Centromeric silencing is relieved in ago1-, dcr1-, and rdp1- mutant strains compared to wild type.

Diagram of the three S. pombe centromeres (A) including locations of ura4+ transgenes as well as outermost (otr), innermost (imr), and central (cnt) centromeric regions. Conserved dg (green) and dh (red) repeats are indicated as arrows. Regions containing one or more tRNA genes are indicated by yellow boxes. Northern analysis (B) of RNA transcripts transcribed from centromeric ura4+ transgenes and a ura4+ (DS/E) mini-gene located on the chromosome arm. Transcripts derived from centromeric repeats were detected by Northern blotting (C and D) using probes specific for dg centromeric repeats

The RNAi machinery is required for the initiation and maintenance of the heterochromatic state of centromeric

repeats.

Reverse strand centromeric transcription occurs in wild-type cells and is degraded posttranscriptionally by the RNAi machinery. Low-level transcription from the

forward strand and/or amplification by Rdp1 results in generation of dsRNA, which is converted to siRNA by RNAi. Rdp1, bound to the chromatin, promotes targeting of histone modifications to specific sequences via siRNA, resulting in maintenance

of the heterochromatic state (HMT, histone methyl transferase).

Modifications of histone H3. Lysine residues on histone H3 can be mono-, di- or tri-methylated. Shown are modifications H3K4me1, H3K4me3 and H3K36me3, which mark active/poised enhancers, active/poised promoters and actively transcribed regions, respectively. me, methylation.At least six mammalian homologs of COMPASS exist, including MLL1–4 complexes, hSET1A and hSET1B, and their recruitment to active promoters can result in H3K4me3

markers of heterochromatin in most eukaryotes

• histone H3 methylated at lysine 9 (H3K9me)

• Su(VAR)2-5 - HP1 - (Swi6 in S.pombe) binds H3K9me

• SU(VAR)3-9 -histone H3-K9 methyltransferase HMTase (Clr4 in S.pombe - Ezh2and G9A in mammals)

nuc nucnucnucnuc

swi6swi6swi6

clr4

nuc

Propagation of heterochromatin platform for the recruitment of other repression activities

H3K9me

Transcriptional regulation through H3K27 methylation. (A) Transcriptional repression regulated by the polycomb repressive complex 2 (PRC2) including EZH1 or EZH2. PRC2 complexes, which include EED, SUZ12, RbAP46/48 and EZH2, catalyze H3K27 di- and tri-methylation. This in turn leads to a more condensed chromatin state and transcriptional repression. PRC2 complexes containing EZH1 can also catalyze H3K27 methylation, or compact chromatin through other mechanism. G9a has H3K27 methyltransferase activity and also affects gene silencing. (B) Transcriptional activation regulated by UTX and JMJD3. UTX and JMJD3 are demethylases that form complexes with MLL, RbBP5 and WDR5. The removal of methyl groups from H3K27me3 leads to transcriptionally active

Active genes are frequently marked by H3K4me3 (see page 407); this modification is presumably removed by LSD1 (which has not yet been characterized in Drosophila). H3K9 is normally acetylated in euchromatin, and this modification must be removed by a histone deacetylase, typically HDAC1. Phosphorylation of H3S10 can interfere with the methylation of H3K9; its dephosphorylation might involve a phosphatase targeted through the carboxy terminus of the protein kinase JIL1 (ref. 10). These transitions set the stage for acquisition of the modifications that are associated with silencing: these include the methylation of H3K9 by SU(VAR)3-9 or another histone methyltransferase, the binding of HP1, and the subsequent methylation of H4K20 by SUV4-20 (an enzyme that is recruited by HP1). Other silencing marks such as methylation of H3K27 by E(Z) (enhancer of zeste; not shown) seem to be relevant in some regions, although this mark is more prominently used by the Polycomb system. Supporting data come from genetic identification of modifiers of PEV, as well as biochemical characterization of the activities of such modifiers and tests of protein–protein interactions10. (Figure adapted, with permission, from ref. 10.)

dsRNA

siRNA

RITS recruitment

RDRC recruitment

dsRNA synthesisDCR1

Swi6-dependent chromatin formation

Clr4

nuc nuc

nuc nuc nuc

RDRC

active RITS

AM

PLI

FIC

AT

ION

Chromatin silencing

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swi6swi6swi6

clr4

nuc

S.pombe

Repeats andtransposons

H3K9me

Methyl transf

RNAi factors such as RITS, RDRC and Dicer, involved in processing of repeat transcripts (red line) into siRNAs, are required for targeting of ClrC to the heterochromatic repeats. siRNA-bound Ago1 is likely to specify the targeting of RITS to nascent repeat transcripts. RITS then facilitates ClrC loading. Rik1 might also directly associate with the repeat transcript and/or some part of the elongating RNAPII complex, thus promoting ClrC loading to nucleate heterochromatin. After the initial methylation of H3K9 by ClrC, Clr4 bound to H3K9me could modify adjacent nucleosomes creating additional binding sites for ClrC and other chromodomain proteins including Swi6 and Chp2 (HPs), which in turn mediate recruitment of factors such as SHREC, thereby promoting higher-order chromatin organization. Swi6 could further contribute to long-range heterochromatin spreading by promoting higher-order chromatin organization by forming oligomers and stabilizing the ClrC binding to chromatin. Boundary DNA elements block inappropriate spreading of heterochromatin into euchromatic regions. Green flag, histone acetylation; red lollipops, H3K9me.RNA dependent RNA polymerase complex - RDRC promote double-stranded RNA (dsRNA) synthesis

Marc Buhler – Basel

Nat Struct Mol Biol. 2013 Aug;20(8):994-1000ncRNA can act as EVICTORS

ncRNAs can counteract the spreading of heterochromatin into neighboring euchromatin

BORDERLINE prevents spreading of the HP1 protein Swi6 and histone H3 Lys9 methylation beyond the pericentromeric repeat region of S.pombeIt acts in a locus-dependent manner and isprocessed by Dicer into sRNAsIt evicts HP1 from the chromatin

Borderline acts as an allosteric regulator – Mass spec diff. interctors if SWI-6 alone or bound to the ncRNA

Role of non coding RNAs and RNAi inDNA methylation and histone modification

Role of non coding RNAs and RNAi inDNA methylation and histone modification

In plants two different RNA polymerase (IV and V)are required for heterochromatin assembly

and for transposon silencing

Pol IV transcription of target loci

with RDR2 and DCL3 24 nt long siRNA AGO4

DRM2 and Pol V +

DNA methylation

RNA dep-RNA pol

methyltransferase

dicer

Pol IVa and Pol IVb are players in the RdDM pathway

RNA polymerase IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3

The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.

higher plants have five multi-subunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability; plus two non-essential polymerases, Pol IVa and Pol Ivb which specialize in small RNA-mediated gene silencing pathways

In the RNA-directed DNA methylation (RdDM) pathway of transcriptional gene silencing double-stranded RNAs generated with the involvement of RDR2 are cleaved by DCL3, and the resulting siRNAs are loaded into AGO4–RISC and/or AGO6–RISC complexes that mediate the de novo methylation of cytosines within DNA sequences complementary to the siRNAs

RNA silencing and DNA methylation in Arabidopsis

• Cytosine DNA methylation silences harmful DNAs such as transposons and retroviruses

• Maintenance DNA methyltransferases propagate pre-existing DNA methylation in the CG sequence context by methylating hemi-methylated sites after DNA replication

• RNA Silencing Genes Control de Novo DNA Methylation in Arabidopsis

Gullerova M, Proudfoot NJ.Cell 2008 132:983-95.

RNAi participates in gene regulation also in euchromatin

HP1 recruits cohesin the protein complex responsible for sister chromatid cohesion during cell division

cohesin is believed to promote transcription termination between

convergent genes -

cohesin

miRNA

siRNAmiRNA

AAA7mG

7mG AAA

P

Taglio dell’RNA

Repressione traduzionale

rasiRNA nuc nuc

Modificazione della cromatina

Figura 3

RNA silencing pathways in different organisms. Long dsRNA and miRNA precursors are processed to siRNA/miRNA duplexes by the RNase-III-like enzyme Dicer. The short dsRNAs are subsequently unwound and assembled into effector complexes: RISC, RITS (RNA-induced transcriptional silencing) or miRNP. RISC mediates mRNA-target degradation, miRNPs guide translational repression of target mRNAs, and the RITS complex guides the condensation of heterochromatin. In animals, siRNAs guide cleavage of complementary target RNAs, whereas miRNAs mediate translational repression of mRNA targets. rasiRNAs guide chromatin modification. S. pombe, C. elegans and mammals carry only one Dicer gene. In D. melanogaster and A. thaliana, specialized Dicer or DLC proteins preferentially process long dsRNA or miRNA precursors. 7mG, 7-methyl guanine; AAAA, poly-adenosine tail; Me, methyl group; P, 5' phosphate.

miRNA in plants miRNA in animals