Solution Structure Of Human Brg1 Bromodomain And Its Specific Binding To Acetylated Histone Tails

This dissertation reports on the solution structure and the acetylated histone binding property of the bromodomain from human Brg1, the core catalytic subunit of the chromatin remodeling complex SWI/SNF.The first part of the dissertation is a substantive review of chromatin remodeling complex (chapter 1), histone code (chapter 2) and the biological function of Brg1 (chapter 3). Chromatin remodeling complexes are coregulators in eukaryotic gene expression. They remodel nucleosomes to expose the promoter of particular genes, enabling the assembly of transcription activators or transcription initiation complexes. Chromatin remodeling complexes can be classified into four groups: SWI/SNF, ISWI, Mi-2 and DOMINO. They are all ATP dependent chromatin modifiers functioning in DNA replication, DNA repair and transcription activation/inhibition.The histone code hypothesis refers to the sequential modification of histone tails including acetylation, methylation, phosphorylation, sumolyzation and ubiqitination. Defined patterns of modifications, possibly acting in combination, can be recognized by specific factors such as bromodomain proteins and chromodomain proteins, which in turn, induce the recruitment of particular effectors , thereby translating the histone-modification pattern into a particular chromatin state to bring about downstream biological reactions including chromatin condensation, DNA repair or transcription activation/inhibition. Histone code is an important content of epigenetic.As the core catalytic subunit of SWI/SNF complex, Brg1 play a critical role in gene regulation, cell cycle control, tumorigenesis, cell proliferation and differentiation. About 5-6% of yeast genes are regulated by Brg1; at least more than 100 genes are affected by Brg1 in mammals. Brg1 can inhibit the expression of E2F target genes including Cyclin E, Cyclin A and CDC2 by up-regulating the expression of p21 and p15, and by interaction with pRB, leading to cell cycle arrest. Therefore, loss of Brg1 function may promote tumor development. 10% of lung cancers are accompanied with loss of Brg1; high ratio of BRG1 mutations are also found in cell lines from breast cancer, pancreas caner and prostate cancer. Many development regulators function by interacting with SWI/SNF complex. SWI/SNF complex involves in the activation of regulatory genes for the development of heamatopoitic cell, myeloid cell, adipose cell, neural cell and myocyte. For example, MyoD recruits Brg1 to the myogenin promoter; the proneural activities of Ngnr1 and NeuroD are Brg1 dependent.The second part of the dissertation details the solution structure of Brg1 Bromodomain and its interaction with acetylated histone peptides. Bromodomain proteins largely associate with chromatin regulation. They recognize histones acetylated at specific lysine residues in deciphering the histone code. It has about 110 amino acids and adopts a left-handed four-helix bundle topology.We expressed ¹⁵N and/or ¹⁵N/¹³C labled Brg1 Bromodomain and obtained purified samples by Ni⁺ affinity chromatography and size-exclusion chromatography. Using 3D NMR methodology, we resolved the solution structure of Brg1 Bromodomain, which conserves the left-handed four-helix bundle topology; on top of which resides the hydrophobic acetyl-lysine binding pocket. The distinct feature of Brg1 Bromodomain is that itsαZ helix is about 4 residues shorter than published bromodomain structures. Wefound it shares the highest structural similarity with scGCN5 (RMSD≈2.1 A).Using NMR perturbation studies, we demonstrate that Brg1 Bromodomain binds acetyl-lysine in the context of histone tails, with no comparable affinity for unacetylated peptides. The bindings are generally weak. The estimated dissociation constants (K_D) for acetylated histone peptides H4-AcK8 and H4-AcK12 are 4.0 mM and 3.6 mM respectively. The dominant substrate was H3-AcK14 (K_D≈1.2mM).Using Molecular Dynamics (MD) simulation with the crystal structure of scGCN5-H4-AcK16 (PDB ID 1E6I) complex as a template, we built a model of the Brg1 Bromodomain in complex with H3-AcK14. The model reveals that residues F1485, L1488, L1494, F1539 and N1540 are interacting with H3-AcK14 peptide; and the backbone carbonyl of T1538 forms a hydrogen bond with the side-chain NH₂ of Arg 17 of H3-AcK14.To verify the interactions indicated by NMR titration and the complex model, we performed mutagenesis research both in Brg1 Bromodomain and H3-AcK14. We obtained three Brg1 Bromodomain mutants (V1484/A, F1539/A, N1540/A) and one H3-AcK14 mutant (R17/A), with which NMR titration experiments were carried out. The mutagenesis analysis confirms that F1539 and N1540 are essential for H3-AcK14 binding, while V1484 is irrelevant, and that the hydrogen bond between T1538 and Arg 17 of H3-AcK14 is critical for H3-AcK14 selectivity.