Structural and Biochemical Mechanisms of MLL1 Activation on Chromatin
dc.contributor.author | Ayoub, Alex | |
dc.date.accessioned | 2022-05-25T15:25:35Z | |
dc.date.available | 2022-05-25T15:25:35Z | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022 | |
dc.identifier.uri | https://hdl.handle.net/2027.42/172670 | |
dc.description.abstract | Transcription is a critical process by which cells regulate temporal and spatial expression of genes. In eukaryotes, histone H3 lysine 4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The existence of six functionally distinct MLL/SET1 H3K4 methyltransferase family members further underscores the biochemical complexity of transcriptional regulation. H3K4 methylation levels are highly correlated with transcription activation and are a frequently used histone post-translational modification to predict transcriptional outcome. Cellular rearrangement of the H3K4 mono-methylation landscape at distal enhancers precedes cell fate transition and identifies novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions can predict intrinsic tumor suppression properties of regulatory regions in a variety of cancer models. Understanding the regulatory mechanisms of H3K4 methylation is paramount as dysregulation of these enzymes almost universally results in establishment and/or progression of developmental disorders and malignant transformation in cancers. Therefore, determining how MLL/SET1 members engage and methylate chromatin is central to deconstructing the mechanistic requirements for H3K4 methylation in cells. First, we use structural and biochemical methods to investigate how MLL1 catalytic SET domain (MLL1SET) binds to nucleosome core particles (NCPs), its native substrate. Using single-particle cryo-EM, we show that MLL1 binds near the dyad axis through ASH2L and RbBP5 binding motifs, the majority with nucleosomal DNA. We show loss of these motifs attenuate MLL1SET catalysis in vitro in an NCP-specific fashion underscoring their importance in MLL1SET engaging chromatin. Next, we use advanced NMR and cellular work to show that central to this MLL1SET-NCP interaction is DPY30. We show that MLL1SET is an NCP compared with recombinant H3. We reveal that DPY30 binding ASH2L is central to this effect, functions universally amongst MLL/SET family members. We show that DPY30 induces drastic changes in ASH2L intrinsically disordered regions (IDRs) resulting in newly resolved resonances. We show loss of any ASH2L IDR attenuates DPY30-mediated stimulation and removal of DPY30 induces immense MLL1 rotational dynamics on the NCP interface with RbBP5 as the sole anchor and overall instability in the ASH2L arm. Lastly, we show that de novo H3K4me3 in cells depends strictly on DPY30. Finally, we use cryo-EM to show that MLL1SET complex engages with NCP in a dynamic interplay of two discrete interaction modes. Using a catalytically inert H3 K4-to-M NCP (NCPK4M), we readily capture these distinct states. Specifically, we show biochemically that interaction motifs found in this alternative mode do not strongly affect MLL1SET methylation or binding in an NCP-specific manner. Despite overall strong structural agreement with ySET1-NCP, our results suggest MLL1SET-NCP regulation occurs divergently through unique rotational dynamics on the NCP interface with ASH2L acting as an anchor. We provide several new aspects of MLL1SET regulation on the nucleosome. Our structural findings reveal new insights into recognition and activation of MLL1SET complex on the NCP. We provide new evidence for divergent regulation of MLL1 through rotational dynamics distinct from ySET from which MLL. Additionally, unique from ySET1, we show critical functions for conserved subunit DPY30. We revealed novel roles in MLL1SET activation on NCP through IDRs and complex stability, previously unknown. This thesis provides mechanistic insights into a completely novel mechanism of methyltransferase regulation through IDRs. Because IDRs exist abundantly in many epigenetic proteins, these findings provide foundational evidence for future investigations into IDRs. | |
dc.language.iso | en_US | |
dc.subject | Eukaryotic transcriptional regulation | |
dc.subject | Mixed Lineage Leukemia | |
dc.subject | cryo-EM | |
dc.subject | H3K4 methylation | |
dc.subject | histone post translational modification | |
dc.subject | KMT2 | |
dc.title | Structural and Biochemical Mechanisms of MLL1 Activation on Chromatin | |
dc.type | Thesis | |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Chemical Biology | |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | |
dc.contributor.committeemember | Dou, Yali | |
dc.contributor.committeemember | Mapp, Anna K | |
dc.contributor.committeemember | Cho, Uhn-Soo | |
dc.contributor.committeemember | Dressler, Gregory R | |
dc.contributor.committeemember | Su, Min | |
dc.subject.hlbsecondlevel | Biological Chemistry | |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | |
dc.subject.hlbtoplevel | Health Sciences | |
dc.subject.hlbtoplevel | Science | |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/172670/1/bouya_1.pdf | |
dc.identifier.doi | https://dx.doi.org/10.7302/4699 | |
dc.identifier.orcid | 0000-0003-0341-8747 | |
dc.identifier.name-orcid | Ayoub, Alex; 0000-0003-0341-8747 | en_US |
dc.working.doi | 10.7302/4699 | en |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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