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Role of Fun30 remodelers in retrotransposon regulation [ChIP-chip and expression analysis]

ABSTRACT: Retrotransposable elements are genomic parasites that are both key drivers of evolution and the ancestors of retroviruses. Although host cells have developed numerous mechanisms to keep these elements in check, there is also evidence from yeast to mammals that stressed cells sometimes activate transposition. Here we show that the fission yeast tf2 retrotransposons are regulated by alternative transcription start site (TSS) usage. Which TSS is used is determined by nucleosome positioning, which is in turn controlled by the Fun30 chromatin remodelers Fft2 and Fft3. By maintaining a high level of nucleosome occupancy at retrotransposon-flanking Long Terminal Repeat (LTR) elements, Fft2 and Fft3 promote the use of a downstream TSS and the production of RNA incapable of reverse transcription and retrotransposition. More generally, we show that Fft2 and Fft3 are involved in repressing the transcriptional response to stress, of which retrotransposon activation is a part. Finally, we show that in stressed cells retrotransposon TSS usage switches, allowing the production of full-length retrotransposon RNA. We propose that allowing retrotransposon transcription from a nonproductive TSS allows for the rapid activation of these elements in times of stress, while preventing their uncontrolled proliferation in the genome.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE57068 | GEO | 2016/03/03

SECONDARY ACCESSION(S): PRJNA245876

REPOSITORIES: GEO

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Project description:Purpose: The ultimate goal of this study is the identification of transcription elongation-related chromatin remodeler and its in vivo mechanism in fission yeast through Next-generation sequencing (NGS) based ChIP-seq technique. Methods: Every ChIP-seq cells were grown up to mid-log phase and harvested after final 1% formaldehyde fixation and 125mM glycine quencing. Cells were harvested by centrifugation at 4 Celcius degrees and the cell pellets were washed by 15mL of pH7.5 TBS buffer per a conical tube. ChIP cells were disrupted by glass bead vortexing in ChIP lysis buffer and sonicated by Sonic Dismembrator Model 500, Fisher Scientific. Debris of the sonicate was excluded by centrifugation by 14,800rpm at 4 Celcius degree. IP samples were aliqouted from the sonicate and immunoprecipiated by proper amount of antibody and protein A/G beads, washed by ChIP lysis buffer for over-night at 4 Celcius degrees. IP samples were washed and eluted by proper buffers in Sigmaprep spin column. Proteins in both IP and input samples were degraded by 2hr 30ug proteinase K rxn and de-crosslinked at 65 Celcius degrees for at least 8hrs. The IP and input DNA were eluted by Quiagen PCR purification kit. Results: RNAPII and factor ChIP-seq in various mutants of chromatin remodelers showed that FUN30 family promotes transcription at gene coding regions. RNAPII ChIP-seq in fft3Δ, spt16-1 and fft3Δspt16-1 demonstrated that RNAPII regulation function of Fft3 is largely overlapped with that of Spt16. Histone H3 ChIP-seq results in fft3Δ and spt16-18 demonstrated that Fft3 is major chromatin remodeler to promote nucleosome turnover through nucleosome disassembly. Finally, histone H3 and RNAPII ChIP-seq in fft3Δ, H3/H4DD and fft3Δ H3/H4DD were showed that Fft3 facilitates RNAPII elongation at ORF regions through nucleosome barrier regulation. Conclusions: Fun30-Fft3 is major chromatin remodeler to facilitate RNAPII elongation by nucleosome disassembly-driven nucleosome barrier alleviation in fission yeast.

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Role of Fun30 remodelers in retrotransposon regulation [ChIP-chip and expression analysis]

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