1. Swi6/HP1 Recruits a JmjC Domain Protein to Facilitate Transcription of Heterochromatic Repeats 
Martin Zofall, Shiv I.S. Grewal 
Molecular Cell 
Volume 22, Issue 5, Pages (June 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Epe1 Colocalizes with Swi6
(A) Epe1-FLAG and Epe1-GFP proteins are functional. The physical map of the mat locus is shown, including a 20 kb heterochromatin domain encompassing mat2P and mat3M as well as cenH that shares homology to centromeric repeats. Inverted repeats, IR-L and IR-R, serve as heterochromatin barriers. Deletion of IR or Δepe1 causes heterochromatin spreading and silencing of ura4+ gene inserted into a euchromatic location (IRRos::ura4+), measured by serial dilutions of cultures onto nonselective medium (N/S), medium lacking uracil (−URA), or counterselective FOA-containing medium (FOA). Expression of IRRos::ura4+ was not affected in cells in which the wild-type copy of epe1 is replaced with either epe1-FLAG or epe1-GFP.
(B) Colocalization of Epe1 and Swi6. Epe1-GFP and Swi6 were visualized by coimmunofluoresence microscopy with antibodies against GFP and Swi6.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Genome-Wide Profiling of Epe1
(A) Chromosomal distribution profile of Epe1. Schematic representations of three S. pombe chromosomes are shown with heterochromatin regions indicated in red (top). Distributions of Epe1 are plotted (bottom). DNA recovered by chromatin fractions immunoprecipitated with anti-FLAG antibodies from Epe1-FLAG cell extracts or from whole-cell extract (WCE) was amplified, labeled, and hybridized to S. pombe oligo array. Relative enrichments were determined by dividing normalized Cy5-labeled ChIP signal against Cy3-labeled WCE signal (y axis). Chromosome positions correspond to the fully assembled virtual contigs (Sanger Center Pombe Genome Database) (x axis). Enrichment at ura4+ reflects crosshybridization to Kint2::ura4+ present in strain used to perform ChIP. Abbreviations: cen, centromere; mat, mating-type region; and tel, telomere.
(B–D) Epe1 displays a distribution profile similar to Swi6 at all major heterochromatic loci. Distribution profiles of Epe1 and Swi6 at centromeres left subtelomere of chromosome 1 and the mating-type locus are shown.
(E) Conventional ChIP analysis of Epe1 binding at heterochromatic loci. DNA isolated from ChIP and WCE fractions was analyzed by multiplex PCR with primers to amplify ura4+ inserted at the mating-type region (Kint2::ura4+) and control minigene-ura4+ (ura4DS/E) at an endogenous location. Similarly, primers designed to amplify centromeric repeats (dg223) or telomere (E12) sequences and act1 control were used to assay Epe1 binding. Intensity ratios of PCR products in ChIP and WCE lanes were used to calculate relative enrichment values shown (see Noma et al. [2001] for details).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Epe1 Localization at Heterochromatic Loci Requires Swi6
(A) Epe1-GFP foci at nuclear periphery are disrupted in swi6 mutant cells.
(B) Epe1 binding to heterochromatic regions correlates with Swi6 levels at these loci. ChIP analysis was used to compare Epe1 and Swi6 levels at different heterochromatic loci in wild-type, Δclr4, ago1, and swi6− mutant background.
(C) Epe1 interacts with Swi6 in vivo. Extracts prepared from cells expressing FLAG-Epe1 or from a control strain were incubated with anti-FLAG M2 Agarose, and immunoprecipitated fractions were analyzed by Western blotting with anti-Swi6 or anti-FLAG antibodies.
(D) Epe1 interacts with Swi6 in vitro. Immobilized GST-Swi6 or GST alone was used to pull down in vitro-translated Epe1 or Epe1 carrying a point mutation in JmjC domain (Epe1 Y307A).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Heterochromatin-Dependent Recruitment of Epe1 to Meiotic Genes
(A) Localization of Epe1 at meiotic genes ssm4 and mei4 and at low complexity (LC) region requires Swi6 and Clr4. ChIP analysis was performed to measure Epe1-FLAG enrichment. Left, Epe1 and Swi6 distributions as determined by ChIP-chip analysis. Right, Epe1 localization at meiotic genes and LC region is abolished in swi6 and clr4 mutant backgrounds.
(B) Epe1 binding to mcp5 and mcp7 requires heterochromatin components.
(C) Epe1 localization at euchromatic intergenic regions flanking bfr1 and spo20 occurs independently of heterochromatin components.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Epe1 Facilitates Transcription of Heterochromatic Repeats by Promoting Pol II Accessibility
(A and B) The levels of transcripts generated by cenH (A) and centromeric dh repeat (B) were analyzed by reverse-transcription PCR (RT-PCR) using RNA prepared from indicated strains. The same RNA samples were also used to amplify control act1 transcript in the presence and absence of reverse transcriptase.
(C) Loss of Epe1 suppresses defects in heterochromatin assembly at centromeric repeats in Δago1 and Δclr3 mutant cells. ChIP analyses were used to measure Swi6 levels at centromeric repeats in indicated strain backgrounds.
(D) Epe1 is not required for transcription of repeat elements in the absence of heterochromatin. Transcript levels from indicated strains were analyzed by RT-PCR.
(E) Epe1 promotes Pol II accessibility to heterochromatic repeats. Pol II occupancy at cenH element was determined by ChIP using 8WG16 antibody that recognizes the carboxy-terminal domain (CTD) of the largest subunit of Pol II.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Epe1 Modulates H3K9me2 in a Swi6-Depenent Manner
(A) Epe1 overexpression disrupts centromeric silencing. Cells harboring control vector Rep3-FLAG or nmt41-FLAG-Epe1 vector expressing wild-type or mutant (Y307A) Epe1 under the control of inducible nmt1 promoter were grown in thiamine-free media to induce Epe1 expression. Serial dilution plating assays in the presence or absence of FOA were done to assay otr1::ura4+ expression.
(B) Mutation in JmjC domain does not affect Epe1 recruitment to heterochromatin. Cells expressing FLAG-Epe1 or FLAG-Epe1Y307A episomally in otherwise Δepe1 background were used to perform ChIPs.
(C) Overexpression of Epe1 affects H3K9me2 levels at centromeric repeats. ChIP analysis was used to measure levels of Swi6, H3K9me2, or H3K9me3 at centromeric dg repeats or otr1::ura4+.
(D) Swi6 is required for Epe1-induced reduction in H3K9me2 at centromeric repeats. Cells overexpressing Epe1 under the control of nmt1 promoter in wild-type or swi6 backgrounds were used to assay H3K9me2 levels by ChIP.
(E and F) Loss of Epe1 is associated with increase in H3K9me and Swi6 levels at ssm4 gene. Results of ChIP analysis with antibodies to H3K9me2, H3K9me3, and Swi6 are shown.
(G) Epe1 defect promotes Clr4 residency at ssm4 locus. ChIP analysis using anti-FLAG antibody was carried out against the FLAG-Clr4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Epe1 Is Required for Transcription and Barrier Function of IRC Elements
(A) Epe1 is required to relieve heterochromatin-mediated repression of IRC1 and IRC3 transcription. RNA isolated from indicated strains was used to perform RT-PCR analysis of IRC1 and IRC3 transcripts.
(B) Pol II occupancy at IRC1 element is reduced in a Δepe1 strain. ChIP with 8WG16 antibody was used to assay for Pol II localization.
(C) Swi6-dependent and -independent mechanisms cooperate to target Epe1 to IRC1. Map of the left cen1 boundary indicating PCR fragment amplified in ChIP assay is shown (top). Results of Epe1-FLAG ChIP in wild-type (wt) and swi6− mutant strains are shown below.
(D and E) Effects of Δepe1 on spreading of H3K9me2 and Swi6 beyond natural heterochromatin borders at cen1 and cen3 loci. Schematic representation of left cen1 and cen3 boundaries indicating PCR fragments amplified in ChIPs is shown (top). Results of ChIP analysis are shown below.
(F) Model for maintenance of heterochromatin. H3K9me targeted by DNA binding factors or RNAi pathway recruits and tethers chromodomain proteins such as Chp1, Chp2, and Swi6, which mediate targeting of distinct activities involved in heterochromatin maintenance. As a component of RITS, Chp1 targets RNAi silencing machinery to heterochromatic regions. Similarly, Swi6 and Chp2 recruit Clr3 HDAC, which represses transcription. In addition, Swi6 targets Epe1 to facilitate transcription of repeats. Balance between these opposing activities is essential to maintain heterochromatin.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions