H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity

The Effects of Histone H2B ubiquitylations and H3K79me3on Transcription Elongation

“…Since then, it has been shown that H2BK34ub nucleosomes have the ubiquitin moieties protrude between the two gyres of the nucleosomal DNA, weakening the structural integrity of the nucleosome and distorting DNA toward the entry region of the nucleosome 20,21 . These results suggest that the effects of H2BK34ub on transcription kinetics can be the major mechanisms of facilitated transcription by H2B ubiquitylations.…”

“…For example, histone methylations via aminoethylation of cystine have been published to successfully reproduce their biochemical functions 35,54,55 . A disulfide coupling approach for ubiquitylation has also been published to reproduce the functions of ubiquitylated histones 21,27,38 . We employed these approaches and a highly refined in vitro transcription system to construct a tractable single-molecule transcription system to investigate the effects of histone H2B ubiquitylations and H3K79me 3 on the kinetics of transcription elongation through the nucleosome and the status of DNA wrapping during and after transcription 43 .…”

“…The interactions involve a general transcription elongation factor Paf1C and the two ubiquitin ligase complexes MOF-MSL and RNF20/40 for ubiquitylating H2BK34 and H2BK120, respectively, which eventually promote Pol II processivity in vivo 16 . Since then, it has been shown that H2BK34ub nucleosomes have the ubiquitin moieties protrude between the two gyres of the nucleosomal DNA, weakening the structural integrity of the nucleosome and distorting DNA toward the entry region of the nucleosome 20,21 . These results suggest that the effects of H2BK34ub on transcription kinetics can be the major mechanisms of facilitated transcription by H2B ubiquitylations.…”

“…An in vitro study has found that H2BK34 ubiquitylation (H2BK34ub) destabilizes H2A-H2B dimer association with the nucleosome, thus facilitating hexasome formation by histone chaperone Nap1 [17][18][19] . Recent Cryo-EM studies of H2BK34 nucleosomes reported that the ubiquitin moieties protrude the DNA gyre gap in the nucleosome and distort the DNA near the nucleosome entry region, thereby destabilizing the nucleosome and facilitating the binding of a histone methyltransferase Dot1L 20,21 . However, no report is currently available to show how such effects of H2BK34ub directly regulate the kinetics of transcription elongation by Pol II.…”

Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes

“…Different histone post-translational modifications (PTMs) (e.g., methylation, acetylation, phosphorylation, and ubiquitination) work together in a combinatorial fashion to alter the nucleosome structure or interact with the chromatin effector proteins in many chromatin-templated processes, including gene silencing, transcription, and DNA damage repair. , Although cell biology and genetic studies can reveal the functional contributions of different PTMs in various epigenetic events and whether they are synergistic or antagonistic, quantitative deciphering of the effect of each PTM pattern necessitates the use of chemical biology methods that enable their biochemical reconstitution in a chemically defined manner. − For instance, in a recent study on the regulatory mechanism of the histone methyltransferase Clr4 during heterochromatin formation, − an intein-based approach was used to prepare K14-ubiquitinated histone H3 (i.e., H3K14ub) to quantify the effect of ubiquitination on Clr4 activity . Meanwhile, to elucidate the activity and selectivity of different HDAC (histone deacetylase) complexes at the nucleosome level, a sortase-based approach was developed to make histones bearing site-specific modifications (e.g., H2BK11/12/20/46ac and H3K9/14/18/23/27ac). , Similarly, in our studies of the recruitment of p53-binding protein 1 (53BP1) in response to DNA double-strand breaks (DSBs), we used total chemical synthesis to make di-ubiquitinated histones and discovered that 53BP1 is a potential reader of both H2AK15 mono-ubiquitination and H2AK13 poly-ubiquitination. , In this context, we now report the synthesis of ubiquitinated and phosphorylated histone variant H2AX through an expedient semisynthetic strategy, integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation.…”