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Molecular mechanism of co-transcriptional H3K36 methylation by SETD2

ABSTRACT: H3K36me3 is a hallmark of actively and recently transcribed genes and contributes to cellular memory and identity. The deposition of H3K36me3 occurs co-transcriptionally when the methyltransferase SETD2 associates with RNA polymerase II. Using electron microscopy assisted by protein-protein crosslinking mass spectrometry, we solved three cryo-EM structures of SETD2 bound to Pol II elongation complexes at different states of nucleosome passage. Together with functional probing, our results suggest a 3-step mechanism of transcription-coupled H3K36me3 deposition. First, binding to the elongation factor SPT6 tethers the catalytic SET domain in proximity to the upstream DNA. Second, Pol II nucleosome passage leads to the transfer of a hexasome from downstream to upstream, poised for methylation. Finally, continued transcription leads to upstream nucleosome reassembly, partial dissociation of the histone chaperone FACT and sequential methylation of both H3 tails, completing H3K36me3 deposition of an upstream nucleosome after RNA polymerase II passage.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Olexandr Dybkov

LAB HEAD: Henning Urlaub

PROVIDER: PXD067197 | Pride | 2025-09-18

REPOSITORIES: Pride

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			Action	DRS
	JWalshe_ECs64NCP64_Try_Olex_UP.fasta	Fasta
	J_Walshe_pLink_reports.zip	Other
	ODybkov_JWalshe_140323_270323_Expl2_ECstarNCP64_BS3_bRP_i_1.raw	Raw
	ODybkov_JWalshe_140323_270323_Expl2_ECstarNCP64_BS3_bRP_i_2.raw	Raw
	ODybkov_JWalshe_140323_270323_Expl2_ECstarNCP64_BS3_bRP_i_3.raw	Raw

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Project description:Maintenance of the correct level and organization of nucleosomes is crucial for genome function. Here we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in maintenance of nucleosome architecture in fission yeast. Cells lacking abo1+ experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide. A chromatin-seq/MNase-seq approach called Chromatin Particle Spectrum Analysis (Kent et al., (2011) Nucleic Acids Res. 39:e26) was used to map and compare nucleosome position in wild-type (strain 972) and isogenic abo1 knock-out (strain HM463) fission yeast cells. For each strain, three independent in vivo MNase digest bio-reps were performed and the purified DNA pooled. CPSA was performed using paired-end mode Illumina technology with pooled samples multiplexed over two HiSeq2000 lanes. Each CPSA paired read describes a microcococcal nuclease (MNase) resistant DNA species from chromatin, with the insert-size equivalent to the size of DNA protection. For each strain type, two analysed data sets are provided here: one listing the genomic distribution of MNase-protected DNAs of 150bp (â150bp CPSA size classâ) and corresponding to mono-nucleosomes; the other listing the genomic distribution of MNase-protected DNAs of 300bp (â300bp CPSA size classâ) and corresponding to di-nucleosomes.

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Molecular mechanism of co-transcriptional H3K36 methylation by SETD2

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