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. Here we present three cryo-EM structures of SETD2 bound to RNA polymerase 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, RNA polymerase 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.

Fig. 1. Upstream H3K36me3 activity and cryo-EM structure of the State 1 poised methylation complex.

a Schematic representation of nucleosome template and proteins used in (b) SETD2 deposits H3K36me3 on an upstream nucleosome. RNA extension by the methylation-competent elongation complex through mononucleosomes with extended run-up distance and T-less cassette to bp 32 (SHL − 4) into the nucleosome. RNA primer contains a 5’ Cy5 label. RNA lengths are indicated on the right side of the denaturing PAGE gel and ssRNA marker on the left. Western blot for H3K36me3 of RNA extension assay products. Assay performed in triplicates. Source data are provided as a Source Data file. c Overall structure of the State 1 complex with magnified view of FACT binding to upstream hexasome. d Alternate view of the State 1complex.

Fig. 2. CXL-MS analysis poised methylation complex.

a Schematic overview of the RTF1 and SPT16 domain architecture. Interchain BS3 crosslinks are visualized between RTF1 and SPT16. Only crosslinks with a -log₁₀Score > 1 are shown. Crosslinks to the N-terminal remnant of the RTF1 and SPT16 affinity tags are not displayed. Initial visualization of crosslinks performed in xiNET. b Magnified view of FACT “fastener” binding SPT16. Interchain BS3 crosslinks are visualized. SPT16 visualized as coulombic surface potential. c Schematic overview of the SPT6 and SETD2 domain architecture. Interchain BS3 crosslinks are visualized between the proteins (d) overall structure of the complex. SPT6 residues that crosslink with SETD2 colored yellow. Magnified view of the SPT6 binding region of SETD2.

Fig. 3. Cryo-EM structure of SETD2 bound to proximal and distal H3 tail.

a SPT16 CTD binds exposed H2A–H2B dimer of unwrapped nucleosome. b 4.84 Å reconstruction of SETD2 bound to proximal H3 tail of the unwrapped upstream nucleosome (c), 3.6 Å reconstruction of SETD2 bound to the distal H3 tail of the unwrapped upstream nucleosome. A gaussian filter with a 2σ standard deviation was applied to the cryo-EM map. d As in (c), without filter (e, f), overall architecture of State 2 and State 3 complexes with SETD2 bound to the proximal and distal H3 tails, of an upstream nucleosome, respectively.

Fig. 4. SPT6 SETD2 interaction required for efficient methylation.

a Schematic overview of the SPT6 showing DLD truncation. b Normalized 3H activity of co-transcriptional methylation assays. c Denaturing PAGE gel of co-transcriptional methylation assays as in (b), with cold S-Adenosyl methionine. Expected nucleosome dyad positions indicated on the right. Undigested DNA/RNA hybrid indicated with *. d Quantification of assay replicates in (c). e Schematic overview of the SETD2 truncations (f), normalized 3H activity of co-transcriptional methylation assays performed with SETD2 or truncations. For data presented in (b, f), each point reflects one replicate (N = 3), depicted as mean ± s.d. Source data are provided as a Source Data file.

Fig. 5. Model for co-transcriptional SPT6 mediated H3K36me3 deposition.

SETD2 auto-inhibition is released by binding both the phosphorylated CTD of RPB1 and the SPT6 DLD of elongating Pol II. Pol II unwraps the downstream nucleosome during passage with SETD2 bound to SPT6, waiting for nucleosome transfer. FACT mediates nucleosome transfer upstream. The binding of the catalytic SET domain to the transferred hexasome is occluded by FACT and is in a poised state. Deposition of the final H2A–H2B dimer allows FACT dissociation from the nucleosome dyad and allows the SET domain to methylate the proximal H3 tail. The unwrapped nucleosome is stabilized by the SPT16 CTD. Further transcription rewraps the proximal histones. SETD2 repositions to the distal H3 tail whilst remaining in proximity to SPT6 DLD.