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. 2015 Dec;16(12):1673-87.
doi: 10.15252/embr.201541214. Epub 2015 Oct 29.

VSports在线直播 - The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast

Laia Sadeghi  1 , Punit Prasad  1 , Karl Ekwall  2 , Amikam Cohen  3 , J Peter Svensson  2
Affiliations

VSports在线直播 - The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast

Laia Sadeghi et al. EMBO Rep. 2015 Dec.

"VSports最新版本" Abstract

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1-Paf1 [a subcomplex of the RNA polymerase II-associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high-resolution genomewide ChIP (ChIP-exo). Loss of Leo1-Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi-independent manner VSports手机版. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover. .

Keywords: Epe1; Paf1C; heterochromatin; histone turnover; transcription V体育安卓版. .

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Figures

Figure 1
Figure 1. Identification of Leo1 in a screen for factors counteracting heterochromatin spreading across the IR‐L boundary

  1. Scheme of the mating type region in Schizosaccharomyces pombe. Integration positions of reporter genes (ura4 + or ade6 +) are indicated V体育平台登录.

  2. Spotting assay using strains with ura4 + and ade6 + integrated as in (A) on plates with added adenine (YEA), with low adenine (YE), or with 5‐fluorotic acid (5FOA).

  3. ChIP–qPCR analysis of H3K9me2 levels at the ura4 + or ade6 + insertion sites depicted in (A). ChIP was performed in three independent experiments (n = 3); error bars indicate the standard deviation (SD) V体育官网入口.

  4. Schematic representation of the protein domains of Leo1; Sp: S VSports在线直播.  pombe; Hs: human. The different colors represent conserved domains: orange: Paf1 interaction; green: conserved, potentially H3 interacting; pink: conserved region of unknown function, potentially RNA binding.

  5. Growth curves of WT cells and cells with different leo1 + alleles (as indicated).

  6. Bright‐field microscopy images of the indicated strains.

Figure EV1
Figure EV1. The transposon mutagenesis system
  1. A

    Cartoon of the Hermes transposon screen.

  2. B

    Southern blot of the Hermes strain. Probes were complementary to the kanMX region (top panel) present in both leo1::Hermes and leo1∆, leading to a band of length 7–8 kb (Hermes‐kanMX, lower panel, upper arrow) and a band of length 4–5 kb (leo1∆::kanMX) (lower panel, lower arrow) VSports app下载.

  3. C–E

    The Ddb1 and epe1::GFP effect on other components of Paf1C. Genetic interaction between ddb1 and cdc73 and tpr1. (C) Spotting assay with ura4 + at its endogenous locus. (D) Spotting assay with strains in ddb1∆ background. (E) Spotting assay in epe1 + ::GFP‐derived strains.

Figure 2
Figure 2. Spreading of heterochromatin across IR‐L is specifically affected by Paf1 and Leo1
  1. A

    Model of Paf1C with its subunits and interactors.

  2. B

    Spotting assay using four strains with ura4 + and ade6 + integrated as in Fig 1A, carrying deletions of genes encoding different subunits of the Paf1 complex (as indicated).

  3. C

    Position variegation effect by Leo1 and Paf1. Spotting assay of strains regrown from –Ura or 5FOA plates with the relevant genotypes indicated.

  4. D

    Immunoblot of H2Bub1 and H4 levels in WT, htb1‐K119R, and leo1∆ cells. Numbers indicate the H2Bub1/H4 ratio for each lane.

  5. E, F

    Growth assay performed on a low‐adenine plate (YE) (E) and a 5FOA‐containing plate (F) using mutants with ura4 + or ade6 + integrated as in Fig 1A. The mutants in this panel lack different factors in the H3K4me (set1∆) or H2Bub1 (rfp1∆, shf1∆, or htb1‐K119R) pathways.

Figure 3
Figure 3. Leo1–Paf1 and Epe1 mediate chromatin anti‐silencing

  1. Scheme of the mat locus in S. pombe. Integration position of ura4 + is indicated (upper panel). Spotting assay on YEA and 5FOA plates (lower panel).

  2. ChIP–qPCR across the XbaI site in the matK domain.

  3. ChIP–qPCR across the pericentric dh repeat of centromere 1.

Data information: (B, C) ChIP was performed in three independent experiments; error bars indicate SD.
Figure 4
Figure 4. Genomewide distribution of heterochromatin in leo1∆ cells

  1. A browser view of ChIP–exo signals in the WT and leo1∆ strains using an antibody against H3K9me2 showing the constitutive heterochromatin domains of chromosome 2 (indicated in red in the lower panel). The ratios of RPKM (reads per kilobase per million mapped reads) over the indicated region are shown. Two independent experiments were performed.

  2. Browser views of H3K9me2 ChIP–exo signals in WT and leo1∆ cells at five facultative heterochromatin islands. The RPKM values are shown for each gene and strain.

  3. ChIP–qPCR of H3K9me2 at Tf2s.

  4. ChIP–qPCR of Swi6 in WT, leo1∆, and leo1::Hermes strains at four loci.

  5. Spotting assay of WT and leo1∆ with ura4 + integrated in the Tf2‐3 locus. The top row shows a WT strain with functional endogenous ura4 +; all other strains have the non‐functional ura4‐D18 allele.

  6. Spotting assay using strains with ura4 + integrated close to the SPAC23H3.14 locus.

  7. ChIP–PCR of H3K9me2 at the SPAC23H3.14 locus. Numbers indicate ratios for the signal SPAC23H13.14/leu1.

Data information: (C, D) ChIP was performed in three independent experiments; error bars indicate SD.
Figure EV2
Figure EV2. Heterochromatin formation over retrotransposable element Tf2 pericentric region of chromosome 1

  1. ChIP–exo for H3K9me2 at Tf2.

  2. Position variegation at Tf2 effect by leo1∆. Spotting assay on –Ura and FOA plates of strains previously grown on –Ura or FOA plates.

  3. Spotting assay of indicated strains on YEA and 5FOA media with ura4 + integrated at the left pericentric region of chromosome 1.

  4. Spotting assay of indicated strains on YEA and 5FOA media with ura4 + integrated at the right pericentric region of chromosome 1.

Figure 5
Figure 5. Leo1 aggravates the effects of siRNA loss

  1. Spotting assay to test TBZ sensitivity of the indicated strains. Tenfold serial dilutions of the indicated cultures were grown on rich medium (YEA) in the presence (17 μg/ml) or absence of TBZ.

  2. Loss of minichromosome Ch16. Two independent experiments were performed; error bars show the range.

  3. Representation of centromere 1 in S. pombe. Integration sites of ura4 + are indicated with a blue vertical arrow (upper panel). Spotting assay of strains with the relevant genotypes indicated (lower panel).

  4. Spotting of strains lacking HP1 (swi6∆) or SHREC (clr3) with ura4 + integrated at the imr1.

  5. Spotting of strains lacking FACT (pob3∆) with ura4 + integrated at the imr1.

Figure 6
Figure 6. Small RNAs generated from heterochromatin are strongly reduced but still present without Leo1

  1. Pie charts illustrating proportions between different sRNA populations in WT and leo1∆.

  2. sRNAs mapped across centromere 3. The number of reads from two independent experiments was normalized to the reads mapping to tRNAs. Red dots in the upper panel indicate positions of tRNAs.

  3. Growth of strains with ade6 + integrated at IR‐L on a YE (low‐adenine) plate.

Figure EV3
Figure EV3. Small RNAs and heterochromatin at telomeres and centromeres
  1. A

    sRNA distribution at tel1L and the centromere of chromosome 1.

  2. B

    sRNA distribution at tel2L, the centromere of chromosome 2, the mat region, and tel2R.

  3. C

    Distribution of sRNA populations in ago1∆.

  4. D–F

    H3K9me2 ChIP–exo over centromere 1 (D), centromere 2 (E), and centromere 3 (F). Green boxes indicate the location of the BORDERLINE transcript 2013 and the IRC3 elements 2008.

  5. G

    Spotting of strains with ura4 + at the euchromatic (inner centromeric) side of tRNA barrier at cen1R.

  6. H, I

    H3K9me2 ChIP–qPCR over the IRC1R locus (H) and the outer repeats (otr2) and central core (cnt2) of centromere 2 (I). Experiments were done in three independent experiments; error bars show the SD.

Data information: (A–F) Two independent experiments were performed, and the profiles show the average.
Figure EV4
Figure EV4. Effect of strain genotype on gene expression (or histone retention) at various chromosomal locations
  1. A, B

    Retrotransposable element Tf2 is silenced by an siRNA‐independent mechanism in leo1∆. (A) Growth of indicated strains, with ade6 + integrated at two locations, on a low‐adenine plate. (B) Distributions of sRNA mapping to the 13 Tf2 retrotransposons. Normalized reads are aligned relative to the transcription start site, sense (top panel) and anti‐sense direction (bottom panel) in WT (blue), leo1∆ (red), and rrp6∆ (green) cells.

  2. C, D

    Histone turnover and the effects of acetyltransferases on heterochromatin formation. (C) ChIP–qPCR using antibody against HA in cells with the RITE construct, using primers for dh1, tf2, matK, rad50 +, and spd1 +. ChIP was performed in two independent experiments. (D) Position variegation effect by Mst2 and Paf1. Spotting assay of strains preselected as being Ura+ or FOAR as indicated.

  3. E

    Spotting assay of strains with ura4 + integrated at matL, and ade6 + at IR‐L.

  4. F

    Spotting assay of strains with ura4 + integrated at matK.

Figure 7
Figure 7. Leo1 mediates histone turnover

  1. Diagram of the RITE system. Green triangles represent LoxP sites.

  2. ChIP of H3‐T7 at silent chromatin regions.

  3. ChIP of H3‐T7 at expressed euchromatic regions transcribed by RNAPII (IRC1R, spd1 +, rad50 +, pyk1 +, and scm3 +) or RNAPIII (tRNA).

  4. Spotting assay using strains with ura4 + at the matK region.

  5. Spotting assay using strains with ade6 + integrated at the IR‐L and ura4 + integrated at matL domains.

  6. Spotting assay using strains with ura4 + at the matK region.

Data information: (B, C) ChIP was performed in three independent experiments; error bars indicate SD.
Figure EV5
Figure EV5. The contribution of H2Bub1 on silencing of the pericentric chromatin

  1. sRNA profile over centromere 3. The number of reads from two independent experiments was normalized to reads mapping to tRNAs. Red dots in top panel indicate positions of tRNAs.

  2. sRNA profile in htb1‐K119R.

  3. Spotting assay of strains with ura4 + integrated at imr1R.

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