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SRX1853799: GSM2204435: H3K9me2_SPY5245; Schizosaccharomyces pombe; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 409,471 spots, 20.5M bases, 11.9Mb downloads

Submitted by: NCBI (GEO)
Study: A transcriptionally permissive histone H3 lysine 9 methylation state enables RNAi-mediated heterochromatin assembly
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Heterochromatic DNA domains play important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. In organisms ranging from fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs transcribed from DNA repeats, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation. H3K9me is in turn required for recruitment of RNAi to chromatin and promotes sRNA generation. However, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations, which allow H3K9me2 catalysis but block the transition to H3K9me3. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS). Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct heterochromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in RNAi-mediated genome defense. Overall design: ChIP-seq analysis of histone modifications and chromatin associated proteins
Sample: H3K9me2_SPY5245
SAMN05263610 • SRS1510772 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP was performed as previously described (Yu et al., 2014). Conditions for fixation and immunoprecipitation (IP) are briefly described here. 50 ml cultures of cells were grown overnight (or 24 hours when 2.5 ug/ml tetracycline was added) in YES to an optical density (OD) of 2 and fixed as follows. For H3K9me2, H3K9me3, and Chp1 ChIP, cell were fixed with 1% formaldehyde for 15 min. For Swi6 and Flag-protein ChIP, we used a modified dual-crosslinking protocol in which cells were first incubated at 18 °C for two hours, resuspended in 5 ml of room temperature PBS, and fixed with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS, Thermo Scientific #21565) for 30 min, followed by addition of formaldehyde to 1% final concentration for another 30 min (Zeng et al., 2006). For Swi6 ChIP in Figure S4, the 2 hr incubation at 18 °C was omitted. For each IP, ~ 2 ug of antibody was pre-incubated with 30 ul of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04-745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Ab18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A. For IP with H3K9me3 antibody, Dynabeads M-280 Streptavidin beads were first incubated with 1 ug of anti-H3K9me3 (Diagenode C15500003), followed by blocking with 5 uM biotin. Sheared chromatin lysate was added to the antibody-bead mixture and incubated for 2 hr at 4 °C on a rotating device. Immunoprecipitated DNA was cleaned up using the Qiagen PCR Purification Kit after reversing cross-links, RNaseA, and proteinase K treatment. Immunoprecipitated DNA was eluted from column with the provided elution buffer (50 ul x 2), subjected to additional shearing in a Qsonica water bath sonicator at 20% amplitude for 15 minutes of total shearing time (each cycle is 15” on + 15” off), followed by vacuum centrifugation to reduce the volume to 30 ul. DNA concentration was measured using Qubit dsDNA HS Kit.1 to 10 ng of immunoprecipitated DNA was used for standard Illumina library construction using barcoded adapters and protocol described previously (Wong et al., 2013). Libraries were pooled and sequenced on the Illumina HiSeq2000 or 2500.
Experiment attributes:
GEO Accession: GSM2204435
Links:
Runs: 1 run, 409,471 spots, 20.5M bases, 11.9Mb
Run# of Spots# of BasesSizePublished
SRR3681253409,47120.5M11.9Mb2017-07-27

ID:
2647821

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