Structural And Functional Study Of Human BRPF2 And BS69

Histones comprise the major protein component of chromatin, the scaffold in which the eukaryotic genome is packaged, and are subject to many types of post-translational modifications (PTMs), especially on their flexible tails. These modifications may constitute a‘histone code’and could be used to manage epigenetic information that helps extend the genetic message beyond DNA sequences. This proposed code, read in part by histone PTM–binding‘effector’modules and their associated complexes, is predicted to define unique functional states of chromatin and/or regulate various chromatin-templated processes. This thesis can be divided into two parts, one about the study on the PHD1 finger and PWWP domain of BRPF2, a scaffold protein of the histone acetyltransferase complex MOZ/MORF; the other about the study on the Bromo-PWWP module of BS69, a transcription corepressor.1、Study on the PHD1 of BRPF2Histone acetyltransferases (HATs) are enzymes that catalyze the transfer of an acetyl group from acetyl-CoA to theε-amino groups of lysine on histones, which results in important regulatory effects on chromatin structure and assembly, and gene expression. HATs are highly diverse and generally form multi-protein complexes. Different HAT complexes are composed of various unique subunits. Combination of these subunits contributes to the unique features of each HAT complex. MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX genes expression and embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (Bromodomain-PHD finger protein 1, 2 or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interaction with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes unmodified histone H3 N-terminal tail (unH3), and report the solution structures of this PHD finger in free and in complex with histone unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallelβ-strand pairing with the PHD1’s two-stranded antiparallelβ-sheet. Binding specificity is determined primarily through recognition of arginine 2 and lysine 4 of unH3 by conserved aspartic acids of PHD1, and threonine 6 of unH3 by a conserved asparagine. ITC or NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac and H3T6ph antagonize the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 is important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on function and regulation of these complexes.2、Study on the tandem Bromo-PWWP domains of BS69BS69 is a multidomain cellular protein containing PHD, Bromo, and PWWP in the N-terminal half, and a MYND in the C-terminus. BS69 could function as a transcription corepressor, and indeed, it could interact with various transcription factors and a set of chromatin remodeling factors. In this study, we determined the crystal structure of the tandem Bromo-PWWP domain and found that these two domains are connected and fixed by a Cys3His-type zinc finger. The Bromo, zinc finger and PWWP domains form a compact structural module, and the relative orientation of these domains is fixed. The N- and C-termini of the tandem domains are on the same side of the molecule, and the potential histone binding pockets of Bromo and PWWP domain are on the other side. The PWWP domain has a pocket formed by F251, W254 and F270. This pocket is highly similar with that of BRPF1, which specifically binds to histone H3K36me3. However, the Bromodomain lacks the conserved Asn corresponding to Asn407 of Gcn5, which is critical for Kac recognition. Instead, Tyr191 sits in this seat. This indicates that this Bromodomain may be unusual even or not bind histones. In fact, we have not found any evidence for the interaction between BS69 and histones.There are multiple histone binding domains in the MOZ complexes and BS69 protein, and their exact roles remains unclear. It’s proposed that histone modifiers such as HAT complexes, function in a stepwise manner(2). First they should receive and interpret the signals (e.g. histone modification patterns), which should be site-specific, and be recruited to the target sites. Then they should be further stabilized on the chromatin template to allow the core protein (e.g. MOZ) work efficiently with their substrates. The presence of multiple histone binding domains in one complex also offers a good solution to balance the requirements between dynamic regulation and stabilizing a complex at a locus after initial recruitment.