Structural And Functional Study Of BAP1/ASXL1, Snu13/U3 SnoRNA And CBP Bromodomain/H3K56ac

Regulation of gene expression is a sophisticated process occuring in multi-temporal, multi-spatial, and multi-hierarchical ways, including epigenetics, and translation. Histone post-translational modification is one of the most important ways of epigenetic regulation, involving the interplay among writer, reader and eraser. BAP1,an important deubiquinating enzyme, can be activated by ASXL1 to erase H2AK119ub1, thus antagonizes the function of PRC1. But how ASXL1 activates BAP1 and how BAP 1 localizes at chromatin remains elusive. CREBBP bromodomain, a typical histone acetylation reader, is proposed to recognize H3K56ac. The molecular mechanism of CREBBP bromodomain interacting with H3K56ac, the context under which the interaction occurs and the role of this recognition are still little known. Apart from histone post-translational modification, the processing and maturation of ribosomal RNA directly influence the assembly of protein translational machine, which is also a vital process of gene regulation. U3 snoRNA belongs to box C/D RNA, which guides 2'-O-methylation of ribosomal RNA and facilitates the processing and maturation of ribosomal RNA. Currently, there is little known about the assembly of U3 snoRNP core complex. This thesis introduce my investigation of gene regulation from three aspects.BAP1 is the only one member of UCHs (Ubiquitin C-terminal Hydrolases) family,localized in nucleus, with basal enzyme activity itself. The ASXL1 DEUBAD domain activates BAP1 to deubiquitinate H2AK119ub1. We expressed and purified the complex of BAP1 with ASXL1 DEUBAD, and got crystal with diffraction resolution at 2.6A. Consistent with previous studies, BAP1 alone favors oligomerization, but in the present of ASXL1 DEUBAD, they form a stable heterodimer. Also, GST pull-down experiments demonstrated that BAP1 binds to intact nucleosome through its highly positive charged C-terminal extension(CTE), but cannot interact with the nucleosomal acidic patch, nucleosomal DNA or free histone, which explained the mechanism by which BAP1/ASXL1 localizes at chromatin.U3 snoRNP consist of U3 snoRNA, snu13, fibrillarin and nop56/58. The assembly of U3 snoRNP is initiated by the interaction between snu12 and U3 box B/C, box C/D,subsequently recruits nop56/58 and fibrillarin. We cloned the gene of snu13, fibrillarin,nop56, nop58, then expressed and purified snu13 and fibrillarin. After transcribing U3,box B/C and box C/D in vitro, we characterized the interaction between snu13 and U3.snu13 binds to box B/C of U3 with low affinity of 2.2?M, which suggests box C/D is necessary for U3 snoRNP assembly. According to previous studies, apo-form box B/C is a loop, but possibly transforms into kink-turn structure in the present of snu13, which is called induced fit. We observed that U3 box B/C can form kink-turn structure in solution based on the special pattern of chemical shift of RNA imino hydrogen. Thus,we proposed that snu13 binds to U3 box B/C through conformation selection but not conformation change.H3K56ac is a unique acetylation modification at nucleosome core region, and plays an important role in various DNA-templated process. CREBBP bromodomain is proposed to be a reader of H3K56ac, but lacks detailed molecular mechanism. Here, in collaboration with Xu Li, we solved the crystal structure of CREBBP bromodomain in complex with H3K56ac peptide. Structural analysis revealed that CREBBP bromodomain recognizes H3K56ac in a similar way to the interaction between other bromodomains and acetyl-lysine. The long side chain of acetyl-lysine extends into a hydrophobic pocket, stabilized by multiple water-mediated hydrogen bond network and a highly conserved asparagine. Intriguingly, H3 aN organizes into a-helix in nucleosome, while appears as an extended structure in our crystal structure. GST pull-down experiments identified that CREBBP bromodomain shows no affinity for H3K56-acetylated recombinant nucleosome, but binds to H3K56-acetylated H3/H4 dimer.These results confirmed the existence of extended conformation of H3 ?N, consistent with previous EPR study that conformation of H3 aN in H3/H4 tetramer is inhomogeneous. CREBBP bromodomain significantly promotes CREBBP HAT-mediated H3K56 acetylation. According to our results, we proposed that CREBBP bromodomain sequesters H3K56-acetylated H3/H4 from HAT and facilitates next cycle of HAT-mediated H3K56 acetylation, subsequently histone chaperon CAF1 accommodates H3K56-acetylated H3/H4 dimer and engages in nucleosome assembly.