~14% of the initial particles contained Set2 and further focused classification improved the Set2 density yielding a map at 3.8 Å (Set2:NCP) (Table 1, Fig. In initial 3D reconstruction, with overall resolution at 3.4 Å, a noisy density representing Set2 domain was present near the DNA entry/exit site of the nucleosome (Supplementary Fig. In the electron micrographs the complex is present in various orientations and in a subset of 2D class averages an additional density was bound to the NCP (Supplementary Fig. To stabilize the complex we have introduced H3K36M mutation 44, 45, recently identified in childhood cancers (Supplementary Fig. To visualize the mechanism of H3K36 methylation, we assembled the complex of Set2 and the mimic of H2B ubiquitinated nucleosome 43, the substrate of Set2 (Supplementary Fig. Histone H3K36me3 and H2BK120 ubiquitination are hallmarks of active chromatin. Our structure reveals the interface between nucleosome and Set2, which is a potential drug target for cancer treatments.Ĭryo-EM structure of Set2 bound to nucleosome Moreover, we show that the AWS domain of Set2 interacts with ubiquitin which promotes H3K36 methylation. These interactions with the nucleosome are required for the specificity toward H3K36 methylation. Our structure shows that Set2 makes extensive interactions with H3 αN, H3 tail, H2A C-terminal tail, and stabilizes DNA in an unwrapped conformation 42. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome core particle (NCP) 41. These findings show that overexpression or mutation of Set2 enzymes or H3K36 promote cancer progression.Īlthough Set2 involvement in essential cellular processes is well described, mechanisms of Set2 mediated H3K36 methylation are poorly understood. Set2 family enzymes are overexpressed in multiple myeloma, neuroblastoma, bladder and breast cancers, and correlate with poor prognosis 3, 37, 38, 39, 40. H3.3K36M mutation traps SETD2 methyltransferase on the substrate which results in genome-wide loss of H3K36 methylation and tumor formation 33, 35. Recently, mutation of H3.3K36 to methionine has been found in chondroblastomas, head and neck squamous cell carcinoma, and colorectal cancer 31, 32, 33, 34, 35, 36. In animals, Set2 family enzymes are essential for maintenance of cell identity 26, 27 and are mutated in many patients with clear cell renal cell carcinoma, acute leukemias, bladder cancer, and glioblastoma 28, 29, 30. Recent data show that Set2 and H3K36me are also involved in DNA damage repair 18, 19, 20, regulation of pre-mRNA splicing 21, 22, chromatin condensation 23, and histone exchange 24, 25. Although deposited by the transcription machinery, H3K36me seems to be a silencing mark that prevents initiation of cryptic transcription within gene bodies 2, 15, 16, 17. H3K36me recruits Rpd3S histone deacetylase complex to restore a repressed chromatin state behind the elongating polymerase 13, 14. Both, H3K36me and H2B K120 ubiquitination are enriched over gene bodies 12. In addition to Set2, RNA Polymerase 2 elongation complex recruits Bre1/Rad6 that ubiquitinate H2B K120 10, 11, indicating possible interplay between these two histone modifications.
Set2 is recruited by phosphorylated C-terminal domain of RNA Polymerase 2 during transcription and catalyzes H3K36 methylation over gene bodies 4, 5, 6, 7, 8, 9. One of the essential histone modifications, histone H3 lysine 36 methylation (H3K36me) is deposited by the Set2 methyltransferase onto the H3 tail and is conserved from yeast to humans 2, 3. Histone modifications are a key player in maintaining genome stability, chromosome segregation, and genome expression 1.
Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases.
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