Role of RNAi machinery in the formation of

Chapter 8

#1 Verdel, A, Jia, S, Gerber, S, Sugiyama, T, Gygi, S, Grewal, SI, Moazed, D (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science, 303:672–676.

#2 Volpe, TA, Kidner, C, Hall, IM, Teng, G, Grewal, SI, Martienssen, RA (2002) Regulation of heterochromatic silencing and H3 -9 by RNAi. Science, 297:1833–1837. Schizosaccharomyces pombe is different than S. cerevisiae and is more similar to and plants Role of RNAi machinery in the formation of heterochromatin Heterochromatin - condensed , silenced chromatin Centromeres - include much heterochromatin Centromeres - One does not observe transcription from material adjacent to the centromeres. Transposon repeats - One does not observe transcription.

In yeast (S. pombe not S. cerevisiae), mutations in Dicer, Argonaute and RdRp cause such transcripts to appear. meH3lys4 - associated with active meH3lys9 - associated with inactive genes (exception is S. cerevisiae). Normally centromeres would have low meH3lys4 and high meH3lys9. Mutants have the opposite.

RdRP found associated with centromere (but called RDRC there). PTGS, TGS, RNAi, miRNA

PTGS = post-transcriptional silencing.

TGS = transcriptional gene silencing.

RNAi = RNA interference. <-- Most commonly used term. Can cause PTGS or TGS. miRNA = microRNA. A form of PTGS. First indication that RNAi and heterochromatin are linked.

Mutations in RNAi machinery (Ago1, Dcr1 or Rdp1) produce phenotypes that are similar to swi6 mutants - centromere disappears, also see appearance of forward and backward larger transcripts from the centromeric region. Side trip - RNAi

What was once considered to be a very specific cellular process is now involved in a great many things. RNAi

RNAi discovered in C. elegans (first animal) while attempting to use antisense RNA in vivo

Control “sense” RNAs also produced suppression of target gene! Craig Mello Andrew Fire sense (and antisense) RNAs were 2006 Nobel Prize in Physiology & Medicine contaminated with dsRNA.

dsRNA was the suppressing agent.

Previously detected in plants but the mechanism was not figured out before Mello and Fire. Function of RNAi

Antiviral - Double stranded RNA is an intermediate in the replication of some RNAi viruses. Suppress transposon activity Suppress expression of repetitive DNA (~1/2 or more of the genome in many organisms) Gene regulation inhibit translation stimulate translation reduce mRNA stability Transcriptional gene silencing Silencing might mean modulation Genome stability - maintenance of heterochromatin Future? probably stimulation of transcription How to evoke RNAi

Inject double stranded RNA Express or inject antisense RNA inside a cell Express a gene that has an inverted repeat. Two promoters that point at one other. Expression of 2 different genes whose mRNAs can base-pair over a short region. Info from Molecular Biology by Weaver 4th edition pg 501-507 dsRNA DICER - RNase III family member ATP Dicer RISC - one of the is SLICER. In ADP+Pi p p Drosophila SLICER is the product of the Argonaute gene. Dicer leaves 2nt 3’ p p overhangs & phosphorylated 5’ ends p RISC - RNA-induced silencing complex

p -Dicer Argonaute has a PAZ and a PIWI domain. 21-23 nt siRNP -R2D2 PIWI PAZ p RISC -Armitrage loading The 2 domains p of Argonaute RISC=RNA- complex induced ATP PIWI domain forms a shape like an RNase H. silencing ADP+Pi complex. RISC p In mice there are 4 Ago genes but only Ago2 Argonaute mRNA Target recognition appears to be SLICER.

Dicer participates in selecting the guide RNA p p Target that is passed on to Argonaute. cleavage mRNA Roles of R2D2 and Armitrage are not clear.

p p Amplification dsRNA mRNA ATP p Dicer NTPs ADP+Pi RdRp (RNA directed RNA polymerase) p p PPi

Dicer leaves 2nt 3’ p p overhangs & phosphorylated 5’ p ends p ATP Dicer p ADP+Pi -Dicer 21-23 nt siRNP p p p -R2D2 p p p p RISC -Armitrage loading p RISC=RNA- complex induced ATP silencing complex. ADP+Pi p RISC Argonaute mRNA Target recognition

p p Target mRNA cleavage

Info from pg508 4th ed Molecular Biology 4th ed. Robert F. Weaver. p p McGraw Hill Publishers Source of special genes micro- special genes RNAs special genes introns or 3’ UTRs

12 One interrelated gene silencing system

siRNA 21-27 n (sizes vary) with a 2 n overhang at the 3’ end.

miRNAs 21-24 n

Some people say that miRNA differs because it is expressed from native genes and used to regulate endogenous genes whereas RNAi is artificial. This distinction is artificial. Why is this new?

If the siRNA’s are so common why were they undetected for so long? We have too many acronyms :(

PTGS - Post-transcriptional Gene Silencing RISC=RNA-induced Silencing Complex, targets mRNA RITS= RNA-induced Transcriptional Silencing RDRC = RNA-dependent RNA polymerase complex Heterochromatin formation Figure 2 pg 105 Epigenetics by Allis, Jenuwein and Reinberg. 2007 CSH press

Clr4 is a H3K9 methylase CLR4 CLR4

Swi6 can bind Clr4

Both can bind H3K9me through their chromodomain Fission yeast centromere Central domain is fundamentally different from other regions. Uses CENP-A (cenH3) This is a histone-like . It replaces H3. Outer heterochromatin is called Clr4-dependent heterochromatin. Outer domain promotes heterochromatin also find homologies between Mat loci and at telomere associated regions. At Mat this homology is called cenH. Promotes Clr4-dependent heterochromatin. Some centromeres have genes. Some tRNA genes are between outer repeat and central kinetochore domain. These prevent the heterochromatin from moving into the central domain.

CLR4 Just like all heterochromatin it tries to spread. Propagation Saccharomyces cerevisiae <--- RECAP OF LAST TOPIC!!! pg 69 Allis et al

Rap1 recruits Sir4 ORC recruits Sir1 Abf1 recruits Sir3 ORC = origin recognition complex pg 72 Common feature of heterochromatin is its spreading from a nucleation site Telomeric repeat is recog by Rap1 and maybe yKu

These two proteins can bind Sir4 proteins

Sir4 binds Sir2 and Sir3

Sir2 is a HDAC that deacetylates H4K16ac & H3k9ac !!!!!

This can then spread through region w/o Rap1 because Sir3 and 4 can bind the Looping can be important for spreading deacetylated H4 & H3 tails which helps the see figure 6 spreading.Saccharomycescerevisiae sir2 produces O-acetyl-ADP-ribose

NAD --> O-acetyl-ADP-ribose which stimulates Sir3 multimerization and Sir3 binding to Sir4 and Sir2 letters to nature cells, corresponding to silent chromatin domains at the nuclear histone H3 only when Lys 9 has been methylated by SUVAR39H1. periphery11, whereas the Clr4-G341D strain shows loss of localiza- In S. pombe, Lys 9 methylase activity is required for transcriptional tion from the nuclear periphery and accumulation of more diffuse repression and HP1 localization. Thus, these data make a direct staining over the nucleolus (Fig. 4c). The Clr4-G341D strain shows correlation between methylated, HP1-bound and tran- a very similar pattern of Swi6 distribution to that of the Clr4D strain scriptionally silent heterochromatin. lacking Clr4 (ref. 5). The speci®city of the Swi6 antibody is The fact that HP1 is associated with the enzyme that methylates demonstrated using a Swi6D strain (Fig. 4c). and `marks' histones for HP1 binding suggests a self-maintenance To con®rm that Swi6 association with silent chromatin is depen- model for how HP1 spreads over chromatin to form heterochro- dent on SET domain function, we performed chromatin immuno- matically repressed regions (Fig. 5). An extension of this model precipitation with anti-Swi6 antibodies (Fig. 4d). Centromeric could also explain how silenced heterochromatin could be passed chromatin, known to be associated with Swi6, was enriched relative on during DNA replication. The SUVAR39H1 methylase, bound to to the control (euchromatin) locus in the immunoprecipitated methylated histones via HP1, could direct the methylation of newly sample compared with the total extract12. In both the clr4-G341D deposited histones. This self-maintaining mechanism would then and clr4¢ strains, enrichment of this centromeric sequence was lost. Thus, a mutation in the Clr4 SET domain that abolishesClr4 (yeast) histone is H3 methylase activity disrupts recruitment of Swi6 to silentcalled chromatin. SUV39H1 Loss of association of Swi6 with centromeres shouldin mammals result in SUV39H1 expression of a normally silent marker gene embedded in centro- methylase Swi6 (yeast) is meric chromatin. Figure 4e shows that this is indeed the case. On HP1 HP1 HP1HP1 indicator plates, wild-type strains silence the centromericHP1 in ade6+ Ac Ac Me Me Me Me marker, which results in red, repressed coloniesmammals13; however, in Ac Ac strains lacking Clr4 (D) or strains defective in Clr4 methylase activity (G341D), this ade6+ gene is clearly expressed,chromodomain resulting in Boundary the formation of white colonies. Collectively theseof HP1 results is a show element that methylase activity of Clr4 is required for theH3K9me recruitment binder of Swi6 to silenced centromeric heterochromatin andNote: for today transcrip- we Active chromatin Spreading of silenced and tional silencing. know that Clr4/ HP1-coated heterochromatin The experiments described here ®t well with existingSUV39H data is also show- a ing that SUVAR39H1 and HP1 colocalize atchromodomain heterochromatic Figure 5 A model of heterochromatic self-maintenance by the SUVAR39H1/HP1 5 sites and interact biochemically . We show that HP1protein. recognizes complex.

PROJECT EARLY SIMPLE MODEL a Peptide pull-downBannister, AJ, Zegerman, P, Partridge, JF, Miska, EA, Thomas, JO, Allshire, RC, Kouzarides, T (2001) Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature, 410:120–124. 100% DATE b CLIENT input CD CSD2001 NAME Lys 4 Lysmeth 9 methLys 4 Lysmeth 9 meth Methylated clr4 wt clr4 G341D H3

12 Swi6: CD CD CSD CSD

H3-K4methH3-K9methH3-K4methH3-K9meth c WT clr4 G341D clr4! swi6!

Anti-Swi6

DAPI

Merged

D clr4 G341D e d WT clr4! ! ! WT clr4 G341clr4 swi6 Cen Control

cen1::ade6+ ade6+ Anti-Swi6: T IP T IP T IP

Figure 4 Swi6 localization is dependent on the methylase activity of the Clr4 SET domain. image of a-tubulin staining (red), DAPI (blue) and Swi6 (green) (bottom) are shown. Scale a, Swi6 chromo domain (CD) or chromo-shadow domain (CSD) were tested for binding to bar, 5 mM. d, Chromatin immunoprecipitation using Swi6 antibodies. T, total extract H3 peptide methylated at either Lys 4, or Lys 9. b, Recombinant Clr4 SET-domain protein input; IP, immunoprecipitated sample. Multiplex PCR assessed enrichment of centromeric (or a mutant version, G341D) were tested for histone methylase activity as described6. (cen) sequences over an euchromatic control in the different strain backgrounds. e, Strains c, Swi6 immunolocalization in strains bearing wild-type clr4+ (WT), a complete deletion of bearing mutations in clr4 and swi6 were tested for their ability to transcriptionally silence clr4 (clr4¢), or a missense mutation in the Clr4 SET domain (clr4 G341D). Swi6 the centromeric ade6+ marker on media with limiting adenine (red colonies, repressed; immunolocalization (top), DAPI staining of chromosomal DNA (middle) and a merged white colonies, expressed). An ade6+ strain (white) provides a control.

NATURE | VOL 410 | 1 MARCH 2001 | www.nature.com © 2001 Macmillan Magazines Ltd 123

COGEDE-864; NO. OF PAGES 8

Different means, same end — heterochromatin formation Reyes-Turcu and Grewal 5

Figure 2

Transcriptional gene Asf1 silencing effectors Clr6 Hip1 SHREC Ac

HP1 HP1 HP1 HP1 Nucleation site Me Ac Me Me Me Me

RNA- or DNA-based recruitment Me Me Me Me HistoneHistone Clr4 H3K9me deacetylation/ nucleosome Clr4 Rik1 occupancy Spreading

Current Opinion in Genetics & Development

Heterochromatin promotes transcriptional silencing through histone deacetylation and changes in nucleosome occupancy. H3K9me that is initially

targeted to nucleation sites by DNA- or RNA-based mechanisms can be spread to surrounding sequences via a process that involves Clr4 binding to

methylated H3K9. HP1 proteins (Chp2 and Swi6) bound to H3K9me provide a recruiting platform for loading of the histone deacetylase complexes

PROJECT

SHREC and Clr6-complex, and the histone chaperones Asf1–HIRA. Clr6 and SHREC have overlapping functions in limiting RNAPII occupancy at

heterochromatic loci [9]. Asf1–HIRA facilitates deacetylation of histones by Clr6 HDAC. Asf1–HIRA and SHREC also promote nucleosome occupancy

and eliminate nucleosome-freeMOREregions RECENT,that are thought to prevent MOREaccess to the COMPLEXtranscriptional machinery and enforce transcriptional gene silencing. Reyes-Turcu FE, Grewal SI (2012) Different means, same end-heterochromatin formation by RNAi and

RNAi-independent RNA processing factors in fission yeast. Curr Opin Genet Dev

which are critical for the assembly of repressive hetero- is that loss of SHREC activities (Clr3 and Mit1) affect

DATE CLIENT

chromatic structures2012[9,41,42]. The oligomerization of REYES-TURCUnucleosome occupancy ET AL.at heterochromatic regions,

HP1 bound to methylated H3K9 provides a mechanism resulting in the appearance of hypersensitive sites

for bridging nucleosomes, which may promote chromatin [42]. Similarly, factors involved in SHREC localization,

condensation [43,71,72]. However, recent studies such as Clr4 and HP1 proteins, prevent the appearance of

suggest that chromatin-associated HP1 proteins also pro- nucleosome-free regions within heterochromatin regions

vide a recruiting platform for repressive chromatin-mod- [74]. SHREC acts in an overlapping manner with Af1-

ifying factors that are required for promoting TGS HIRA because clr3 asf1 double mutant show substantial

[41,42,73,74] (Figure 2). reduction in nucleosome occupancy [73]. These analyses

have revealed a critical aspect of heterochromatic silen-

At least three effector protein complexes that act as cing: specifically that HDACs and histone chaperones

critical determinants of heterochromatic TGS have been associated with the H3K9me-HP1 docking platform func-

identified in genetic and biochemical studies. These tion in promoting nucleosome occupancy and the assem-

include the Snf2-HDAC repressor complex (SHREC), bly of repressive chromatin.

a Clr6 HDAC complex, and the Asf1–HIRA histone

chaperone (Figure 2) [42,56,73]. SHREC (containing Concluding remarks

the HDAC Clr3 and the Snf2 family protein Mit1) associ- Recent studies have yielded the surprising finding that

ates with Chp2 and Swi6, both of which are required the transcription of loci assembled in repressive chro-

for its localization across heterochromatic domains matin is critical for targeting of heterochromatin assem-

[9,42,44]. Swi6 also associates with a Clr6 HDAC com- bly machinery. Since heterochromatin formation occurs

plex and Asf1–HIRA histone chaperone [73]. While at several loci dispersed across the genome, a funda-

these effectors can be recruited to specific loci indepen- mental question remains: what makes these loci prefer-

dent of heterochromatin, their association with HP1 ential targets of heterochromatin modifications? Given

proteins allows them to spread and act broadly across the role of transcription and RNAs in this process, it is

silenced domains [41,73]. likely that special features of target RNAs serve as

signals that are recognized by the heterochromatin

Asf1–HIRA facilitates histone deacetylation by Clr6, machinery. Changes in RNAPII transcription of these

which together with SHREC is essential for hypoacetyla- loci may also provide a mechanical signal that is recog-

tion of histones [73]. However, an important observation nized by heterochromatin factors.

www.sciencedirect.com Current Opinion in Genetics & Development 2011, 22:1–8

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

So far not conceptually different

COGEDE-864; NO. OF PAGES 8 from S. cerevisiae

Different means, same end — heterochromatin formation Reyes-Turcu and Grewal 5 except for ...

Figure 2

Transcriptional gene Asf1 silencing effectors Clr6 Hip1 SHREC Ac

HP1 HP1 HP1 HP1 Nucleation site Me Ac Me Me Me Me

RNA- or DNA-based recruitment Me Me Me Me HistoneHistone Clr4 H3K9me deacetylation/ nucleosome Clr4 Rik1 occupancy Spreading

Current Opinion in Genetics & Development

Heterochromatin promotes transcriptional silencing through histone deacetylation and changes in nucleosome occupancy. H3K9me that is initially

targeted to nucleation sites by DNA- or RNA-based mechanisms can be spread to surrounding sequences via a process that involves Clr4 binding to

methylated H3K9. HP1 proteins (Chp2 and Swi6) bound to H3K9me provide a recruiting platform for loading of the histone deacetylase complexes

SHREC and Clr6-complex, and the histone chaperones Asf1–HIRA. Clr6 and SHREC have overlapping functions in limiting RNAPII occupancy at

heterochromatic loci [9]. Asf1–HIRA facilitates deacetylation of histones by Clr6 HDAC. Asf1–HIRA and SHREC also promote nucleosome occupancy

and eliminate nucleosome-free regions that are thought to prevent access to the transcriptional machinery and enforce transcriptional gene silencing.

which are critical for the assembly of repressive hetero- is that loss of SHREC activities (Clr3 and Mit1) affect

chromatic structures [9,41,42]. The oligomerization of nucleosome occupancy at heterochromatic regions,

HP1 bound to methylated H3K9 provides a mechanism resulting in the appearance of hypersensitive sites

for bridging nucleosomes, which may promote chromatin [42]. Similarly, factors involved in SHREC localization,

condensation [43,71,72]. However, recent studies such as Clr4 and HP1 proteins, prevent the appearance of

suggest that chromatin-associated HP1 proteins also pro- nucleosome-free regions within heterochromatin regions

vide a recruiting platform for repressive chromatin-mod- [74]. SHREC acts in an overlapping manner with Af1-

ifying factors that are required for promoting TGS HIRA because clr3 asf1 double mutant show substantial

[41,42,73,74] (Figure 2). reduction in nucleosome occupancy [73]. These analyses

have revealed a critical aspect of heterochromatic silen-

At least three effector protein complexes that act as cing: specifically that HDACs and histone chaperones

critical determinants of heterochromatic TGS have been associated with the H3K9me-HP1 docking platform func-

identified in genetic and biochemical studies. These tion in promoting nucleosome occupancy and the assem-

include the Snf2-HDAC repressor complex (SHREC), bly of repressive chromatin.

a Clr6 HDAC complex, and the Asf1–HIRA histone

chaperone (Figure 2) [42,56,73]. SHREC (containing Concluding remarks

the HDAC Clr3 and the Snf2 family protein Mit1) associ- Recent studies have yielded the surprising finding that

ates with Chp2 and Swi6, both of which are required the transcription of loci assembled in repressive chro-

for its localization across heterochromatic domains matin is critical for targeting of heterochromatin assem-

[9,42,44]. Swi6 also associates with a Clr6 HDAC com- bly machinery. Since heterochromatin formation occurs

plex and Asf1–HIRA histone chaperone [73]. While at several loci dispersed across the genome, a funda-

these effectors can be recruited to specific loci indepen- mental question remains: what makes these loci prefer-

dent of heterochromatin, their association with HP1 ential targets of heterochromatin modifications? Given

proteins allows them to spread and act broadly across the role of transcription and RNAs in this process, it is

silenced domains [41,73]. likely that special features of target RNAs serve as

signals that are recognized by the heterochromatin

Asf1–HIRA facilitates histone deacetylation by Clr6, machinery. Changes in RNAPII transcription of these

which together with SHREC is essential for hypoacetyla- loci may also provide a mechanical signal that is recog-

tion of histones [73]. However, an important observation nized by heterochromatin factors.

www.sciencedirect.com Current Opinion in Genetics & Development 2011, 22:1–8

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004 Clr4-dependent heterochromatin Repetitive DNA Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac

pg 157 and pg 105 Allis heterochrm around centromeres in S. pombe Repetitive DNA Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Amplification

RDRC RNA directed RNA polymerase complex Rdp1 - RNA-directed RNA polymerase NTPs Rdrp1 Hrr1 ATP PPi Cid12 Hrr1 - RNA helicase Dicer ADP+Pi Cid12 - related to DNA pol p p p & polyA polymerase <-- !!!! p p ATP Notice that a primer is NOT required!!! Dicer ADP+Pi p p p Book says So far only plants and p p p C. elegans have Rdp1.

RITS - RNA-induced initiator of transcriptional gene silencing or p RITS Ago1 RNA-induced transcriptional silencing

Chp1 - chromodomain protein, part of heterochromatin binds MeH3K9

Tas3 - ???

pg 157 and pg 105 Allis heterochrm around centromeres in S. pombe All of this probably happens on a tethered transcript Nucleation

See the book chapter 8 from Epigenetics pg 157 and pg 105 Allis, Jenuwein and Reinberg 2007 heterochrm around centromeres in S. pombe

Also Reyes-Turcu FE, Grewal SI (2012) Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast. Curr Opin Genet Dev

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin Repetitive DNA formation Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac (b)

RDRC siRNA p1 RNA Rd binding Dcr1 transcript protein/ Clr4 Rik1 Mmi1 Now, represent the repeattranscript like this. Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

nucleation site

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

Diagramheterochromatin is adapted includefrom Reyes-Turcuboth RNAi-dependent FE, Grewal SIand (2012)RNAi-independent Different means, samepathways. end- DNA-sequence based mechanisms rely on sequence-specific DNA

heterochromatinbinding factors formationto recruit by RNAichromatin-modifying and RNAi-independentactivities. RNA processing(b) RNA-based factorsheterochromatin in fission nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and yeast.Dicer. CurrGuided Opinby GenetsiRNAs, Dev the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin formation

(b)

RDRC siRNA p1 RNA Rd binding Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

binding factors to recruit chromatin-modifying activities. (b) RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin RITS = RNA-induced transcriptional silencing complexformation

(b) Dcr1 (dicer) produces siRNA from dsRNA that target RITS to the nascent RNA producedRDRC by the repeat of siRNA heterochromatin regionsp1 at centromeres, subtelomeres and RNA Rd binding silent mat loci.Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

p RITS

In bindinga previous factors diagram,to recruit chromatin-modifying RITS was drawnactivities. like(b) this:RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin RITS recruits Clr4 complex and Clr4 methylates H3K9formation to promote heterochromatin assembly. (b)

RDRC siRNA In mammals, Clr4 p1 RNA Rd binding is called Suv39h.Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

Noticebinding factorsthat Chp1to recruit (chromodomainchromatin-modifying protein)activities. (b)bindsRNA-based H3K9me!heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin RITS recruits Clr4 complex and Clr4 methylates H3K9formation to promote heterochromatin assembly. (b)

RDRC siRNA H3K9 must be p1 deacetylated. Clr6 RNA Rd binding Dcr1 probably does this transcript protein/ Clr4 Rik1 (Clr3 and Sir2 Mmi1 transcript might also do it). Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

Noticebinding factorsthat Chp1to recruit (chromodomainchromatin-modifying protein)activities. (b)bindsRNA-based H3K9me!heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin RDRC is recruited (RDRC = RNA dependent RNAformation polymerase complex). (b)

RDRCRDRC siRNA RNA RdRdrp1p1 Hrr1 binding Dcr1 Cid12 transcript protein/ Clr4 Rik1 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

I dobinding not factorsknowto howrecruit RITSchromatin-modifying and Clr4 complexactivities. recruits(b) RNA-based RDRC.heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin formation RDRC synthesizes RNA using the RNA template. (b)

RDRC siRNA RDRC RNA Rdp1 bi Dcr1 transcript nding Rdrp1 protein/ Hrr1 Rik1 Cid12 Clr4 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

binding factors to recruit chromatin-modifying activities. (b) RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin formation Remember Dicer? (b)

RDRC siRNA p1 RNA Rd binding Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Dicer Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

binding factors to recruit chromatin-modifying activities. (b) RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi- Site-specific DNA dependent independent binding proteins

H3K9me

Heterochromatin Dicer produces more triggers that are used to buildformation more RITS. (b)

RDRC siRNA p1 RNA triggers Rd binding Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Dicer Tas3 Exosome Red1 Stc1 Mlo3-TRAMP Chp1 Ago1 A Pla1 A H3K9me A H3K9me A RITS A Rik1ik1 A Me Me A Me A Clr4 ? RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

nucleation site

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

binding factors to recruit chromatin-modifying activities. (b) RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

Current Opinion in Genetics & Development 2011, 22:1–8 www.sciencedirect.com

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004

COGEDE-864; NO. OF PAGES 8

After nucleation weDifferent get means, heterochromatinsame end — heterochromatin formation formationReyes-Turcu and Grewal 5 and spreading that does not involve non-coding

Figure 2 RNAs.

Transcriptional gene Asf1 silencing effectors Clr6 Hip1 SHREC Ac

HP1 HP1 HP1 HP1 Nucleation site Me Ac Me Me Me Me

RNA- or DNA-based recruitment Me Me Me Me HistoneHistone Clr4 H3K9me deacetylation/ nucleosome Clr4 Rik1 occupancy Spreading

Current Opinion in Genetics & Development

Heterochromatin promotes transcriptional silencing through histone deacetylation and changes in nucleosome occupancy. H3K9me that is initially

targeted to nucleationHP1/swi6,sites by DNA- or RNA-based clr4mechanisms andcan chp1be spread to surroundingall bindsequences H3K9me.via a process that involves Clr4 binding to

methylated H3K9. HP1 proteins (Chp2 and Swi6) bound to H3K9me provide a recruiting platform for loading of the histone deacetylase complexes

SHREC and Clr6-complex, and the histone chaperones Asf1–HIRA. Clr6 and SHREC have overlapping functions in limiting RNAPII occupancy at

heterochromatic loci [9]. Asf1–HIRAWhatfacilitates deacetylationtype ofof histones proteinsby Clr6 HDAC. Asf1 are–HIRA and they?SHREC also promote nucleosome occupancy

and eliminate nucleosome-free regions that are thought to prevent access to the transcriptional machinery and enforce transcriptional gene silencing.

which are critical for the assembly of repressive hetero- is that loss of SHREC activities (Clr3 and Mit1) affect

chromatic structures [9,41,42]. The oligomerization of nucleosome occupancy at heterochromatic regions,

HP1 bound to methylated H3K9 provides a mechanism resulting in the appearance of hypersensitive sites

for bridging nucleosomes, which may promote chromatin [42]. Similarly, factors involved in SHREC localization,

condensation [43,71,72]. However, recent studies such as Clr4 and HP1 proteins, prevent the appearance of

suggest that chromatin-associated HP1 proteins also pro- nucleosome-free regions within heterochromatin regions

vide a recruiting platform for repressive chromatin-mod- [74]. SHREC acts in an overlapping manner with Af1-

ifying factors that are required for promoting TGS HIRA because clr3 asf1 double mutant show substantial

[41,42,73,74] (Figure 2). reduction in nucleosome occupancy [73]. These analyses

have revealed a critical aspect of heterochromatic silen-

At least three effector protein complexes that act as cing: specifically that HDACs and histone chaperones

critical determinants of heterochromatic TGS have been associated with the H3K9me-HP1 docking platform func-

identified in genetic and biochemical studies. These tion in promoting nucleosome occupancy and the assem-

include the Snf2-HDAC repressor complex (SHREC), bly of repressive chromatin.

a Clr6 HDAC complex, and the Asf1–HIRA histone

chaperone (Figure 2) [42,56,73]. SHREC (containing Concluding remarks

the HDAC Clr3 and the Snf2 family protein Mit1) associ- Recent studies have yielded the surprising finding that

ates with Chp2 and Swi6, both of which are required the transcription of loci assembled in repressive chro-

for its localization across heterochromatic domains matin is critical for targeting of heterochromatin assem-

[9,42,44]. Swi6 also associates with a Clr6 HDAC com- bly machinery. Since heterochromatin formation occurs

plex and Asf1–HIRA histone chaperone [73]. While at several loci dispersed across the genome, a funda-

these effectors can be recruited to specific loci indepen- mental question remains: what makes these loci prefer-

dent of heterochromatin, their association with HP1 ential targets of heterochromatin modifications? Given

proteins allows them to spread and act broadly across the role of transcription and RNAs in this process, it is

silenced domains [41,73]. likely that special features of target RNAs serve as

signals that are recognized by the heterochromatin

Asf1–HIRA facilitates histone deacetylation by Clr6, machinery. Changes in RNAPII transcription of these

which together with SHREC is essential for hypoacetyla- loci may also provide a mechanical signal that is recog-

tion of histones [73]. However, an important observation nized by heterochromatin factors.

www.sciencedirect.com Current Opinion in Genetics & Development 2011, 22:1–8

Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004 Nucleation reoccurs in S-phase.

Paradox. RNA polymerase II must access heterochromatin in order to maintain it!!!! HP1/Swi6 recruites antisilencing factors (Epe1) that promote some transcription.

Get transcription in both directions across the repeats.

Grewal SI (2010) RNAi-dependent formation of heterochromatin and its diverse functions. Curr Opin Genet Dev 20: 134-141.

COGEDE-864; NO. OF PAGES 8

2 Genome architecture and expression

Figure 1

(a) Transcription and RNA- DNA sequence- based mechanism based mechanism

RNAi- RNAi-Lest Site-specificyou feel DNA disappointed at the simplicity of this dependent independent binding proteins mechanism, I will assuage your discomfort by

H3K9metelling you that S. pombe has islands of

Heterochromatinheterochromatin elsewhere that are small and use a formationNON-RNAi but RNA-dependent mechanism. (b)

RDRC siRNA p1 RNA Rd binding Dcr1 transcript protein/ Clr4 Rik1 Mmi1 transcript Tas3 Exosome Red1 Stc1 Mlo3-TRAMP This mechanism is used to Chp1 Ago1 A Pla1 A H3K9me A H3K9me A silence meiotic genes RITS A Rik1ik1 A Me Me A Me A Clr4 ? during vegetative growth. RNAP II RNAP II repeat element meiotic gene

Current Opinion in Genetics & Development

(a) Heterochromatin nucleation is mediated by RNA-based and DNA sequence-based mechanisms. RNA-based mechanisms to establish

heterochromatin include both RNAi-dependent and RNAi-independent pathways. DNA-sequence based mechanisms rely on sequence-specific DNA

binding factors to recruit chromatin-modifying activities. (b) RNA-based heterochromatin nucleation pathways. The RNAi-dependent pathway (left)

involves RITS, RDRC and Dicer. Guided by siRNAs, the RITS complex localizes to nascent transcripts where it interacts with Stc1, a scaffold that

bridges RITS to Clr4. Clr4 methylates H3K9 to assemble heterochromatin. Repeat transcripts are polyadenylated by Mlo3-associated TRAMP, a factor

that mediates processing of RNAs by the exosome and the RNAi machinery. The RNAi-independent pathway (right) requires the RNA elimination

machinery to assemble facultative heterochromatin at meiotic genes. The RNA binding protein Mmi1, along with factors involved in pre-mRNA 30-end

processing and the RNA elimination machinery, recognize specific meiotic RNAs. Red1, a protein that interacts with the exosome, may form a

specialized complex that recruits Clr4 required for the assembly of heterochromatin at specific meiotic genes.

regulated genes. Another key finding discussed is that transcriptional silencing (RITS) complex. In addition to

heterochromatin can induce transcriptional silencing, at Ago1, RITS consists of the chromodomain protein Chp1

least in part by promoting nucleosome occupancy. and Tas3 [30]. RITS facilitates the localization of Rdp1

[31], a component of the RNA-dependent RNA poly-

RNAi-dependent targeting of heterochromatin merase complex (RDRC) that also contains the polyA

RNAi is a conserved cellular process involved in post- polymerase Cid12 and the putative helicase Hrr1 [32].

transcriptional gene silencing [22,23]. RNAi also plays a RDRC requires Swi6/HP1 for its localization [31], and

key role in the assembly of heterochromatin in several generates dsRNA [31,32] in a process involving polyade-

organisms [1,12,24,25,26]. In S. pombe, the core RNAi nylation of transcripts by Cid12 [32].

factors Argonaute (Ago1), Dicer (Dcr1), and RNA-de-

pendent RNA polymerase (Rdp1) target transcripts siRNAs are believed to target RITS to nascent repeat

generated by dg and dh repeats embedded in major transcripts via interaction with Ago1 (Figure 1b) [30]. RITS

heterochromatin domains at centromeres, subtelomeres localization to heterochromatic loci also depends on the

and the silent mat locus [27,28,29]. Dcr1 processes binding of Chp1 to H3K9me [29,33–35]. RITS processes

double stranded RNA (dsRNA) into small interfering repeat transcripts and mediates the localization of Clr4/

RNAs (siRNAs), which are loaded onto the RNA-induced Suv39h [36], a methyltransferase involved in methylation

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Please cite this article in press as: Reyes-Turcu FE, Grewal SIS. Different means, same end — heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission

yeast, Curr Opin Genet Dev (2012), doi:10.1016/j.gde.2011.12.004