The role of lysine methylation in disease pathogenesis [electronic resource]

Epigenetics is the study of heritable changes in gene expression that occur independent of changes in the primary DNA sequence. Chromatin structure defines the state in which genetic information is organized in the cell. The organization of this structure greatly influences the abilities of genes to be activated or silenced. In eukaryotic cells, 146 base pairs of DNA is wrapped around the histone octamer (two H2A/H2B dimers and one H3/H4 tetramer) forming the nucleosomes, the basic unit of chromatin. The nucleosome cores are connected by linker DNA sequences to further package into higher-order chromatin structures. In addition to the core histones, each histone contains an unstructured N-terminal tail. The histone tails are the sites of most of the post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation. These modifications regulate the structure and function of chromatin through two general mechanisms. In the first model, histone modifications may play a direct role in altering chromatin structure. For example, histone acetylation neutralizes the positive charge of lysine residues and thus, affecting the interactions of the histones with DNA, transcription factors and other nucleosomes. Secondly, histone modifications can indirectly affect chromatin functions by serving as a binding platform for modular proteins and complexes. For instance, the methylation of histone H3 at lysine 9 is recognized specifically by the chromatin organization modifier (Chromo) domain of heterochromatin protein 1 (HP1), which contributes to the induction and the propagation of heterochromatin structure. Ten years ago, Strahl and Allis proposed a general idea of "histone code" hypothesis, which states that histone modifications, distinct or in combination, to form a "code" to influence chromatin structure and lead to varied transcriptional outputs. In recent years, many chromatin regulators were identified, such as the proteins that "write" or "erase" or "read" the modifications. Some chromatin regulators are expressed in a tissue-specific manner and play important roles in physiology and disease pathogenesis. For instance, the H3K27 histone methyltransferase EZH2 is overexpressed in tumors such as prostate, breast, colon, skin and lung cancer. Disruption of normal patterns of covalent histone modifications is another hallmark of cancer. One of the most characterized examples is the global reduction of the trimethylation of H4K20 and acetylation of H4K16, along with hypomethylation, at repeat sequences in many tumors. Since post-translational modifications have been shown to be important for many biological processes such as gene expression, DNA damage and repair and apoptosis, disruption of these processes has been linked to carcinogenesis and other disease pathogenesis. The discovery of reversible mutations in the epigenetic machinery makes post-translational modifications as one of the most promising and expanding fields in the current biomedical research. Methylation does not neutralize the charge of the modified residue nor does addition of methyl groups add considerable bulk, this mark is believed to create a distinct molecular architecture on histones that is recognized by specialized binding domains present within chromatin-regulatory proteins. The proteins and domains that recognize histone modifications, named "effectors" or "readers", are thought to define the functional consequences of lysine methylation by transducing molecular events at chromatin into biological outcomes. Mutations in these "readers" proteins have been shown to link to many disease pathogenesis. However, relatively few effector domains have been identified in comparison to the number of modifications present on histones and non-histone proteins. Here we developed a human epigenome peptide microarray platform (HEMP) for high-throughput discovery of chromatin effectors. We probed this platform with modification-specific antibodies and known chromatin effector domains to test the integrity of the peptides on the slides. We also screened a library of Royal Domain family members and identified three effector proteins with novel modified-histone binding activity. Hence, the development of the HEMP facilitates the identification of effector proteins and understanding of chromatin signaling networks. Multiple Myeloma (MM) is a malignancy of bone marrow plasma cells that frequently results in bone marrow destruction, bone marrow failure and death. 15% of patients with multiple myeloma is diagnosed with an immunoglobin gene, t(4; 14), translocation. MM patients carrying the t(4; 14) translocation is associated with the overexpression of WHSC1/MMSET/NSD2. NSD2 is a protein lysine methylatransferase in the nuclear receptor binding SET domain protein family. However, the molecular mechanism by which NSD2 contributes to myeloma pathogenesis is not known. Here we show that the dimethylation of histone H3 at lysine 36 (H3K36) is the principal physiological activity of NSD2. In mammalian cells, H3K36me2 normally maps to gene bodies. In t(4; 14)+ myeloma cells, overexpression of NSD2 disrupts the physiologic genomic organization of H3K36me2 which is found being dispersed throughout the genome. NSD2 expression is linked to transcription activation and H3K36me2 location at gene bodies positively correlates with transcription levels. In Myeloma cells, NSD2-mediated localized elevation of H3K36me2 induces transcription at normally inert cancer-associated genes. Catalytic activity of NSD2 confers tumor formation in xenograft model and promotes oncogenic transformation of primary cells by regulating transcriptional programs that favor oncogenesis. The BAH domain is an evolutionarily conserved chromatin-associated motif. Utilizing the HEMP, we screened several BAH domains from yeast and human for binding activity. We found that the BAH domain of human ORC1 specifically bind to H4K20me2 peptides. Structural studies show that BAH domain has an aromatic dimethyl-lysine-binding cage that interacts with the bound peptide. ORC1 is dispensable for ORC complex assembly but is necessary for loading of the complex into chromatin. The ability of ORC1 BAH domain binding to H4K20me2 is required for the efficient stabilization of ORC complex at chromatin. H4K20me2 is enriched at replication origins. Abrogation in ORC1 and H4K20me2 interactions impairs cell-cycle progression. Mutations in ORC1 BAH domain have been implicated in aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism. In a zebrafish model, orc1 morphants display an MGS-like dwarfism phenotype, which can be rescued by wild type Orc1 but not ORC1 binding mutants. Zebrafish depleted with H4K20me2 also displays the MGS-like phenotype. Together, our findings reveal a new function for histone methylation signaling at chromatin in the regulation of DNA replication and organismal growth. KDM2A is the first jumonjiC domain-containing demethylase identified. We solved the co-crystal structure of KDM2A and its substrate, H3K36me2. We found that KDM2A demethylation activity is required to maintain genomic stability. We also show that KDM2A is a tumor suppressor and its demethylation activity is required for suppressing cellular transformation.

Keywords: Biology.
Advisor: Gozani Or Pinchas primary advisor. Thesis advisor
Chua Katrin Faye advisor. Thesis advisor
Utz Paul advisor. Thesis advisor