Functional characterization of histone variant H2A.B in Hodgkin lymphoma

Abstract

The replacement of canonical histones with their variant forms is a key process by which all aspects of genome function is regulated. The histone variant H2A.B is a testis-specific epigenetic regulator, with roles in activating gene transcription and modulating mRNA splicing. Testis-specific epigenetic regulators are commonly ectopically expressed in cancer, and often play a pivotal role in oncogenesis. The overexpression of H2A.B in Hodgkin lymphoma (HL) raises the hypothesis that H2A.B plays a role in HL oncogenesis. Hence, the main aims of the thesis were to determine whether H2A.B has acquired a novel role in oncogenesis, and to understand the mechanism of H2A.B-mediated epigenetic regulation in HL. One important regulatory mechanism is to add post-translational modification (PTM) to histones. Whether the function of H2A.B is controlled by PTM was also investigated. Finally, the question of whether the epigenetic regulatory features of H2A.B in the testis are conserved in HL was also addressed. In Chapter 3, H2A.B knock-down inhibited cell proliferation suggesting that H2A.B plays a role in HL oncogenesis. H2A.B is enriched in the nucleoli of both primary tumours and their derived cell lines. Correspondingly, H2A.B enhanced rDNA transcription by potentiating Pol I initiation. Strikingly, H2A.B-containing nucleosomes were also enriched at Pol II transcribed ribosomal protein (RP) genes, which created a more accessible chromatin template that correlated with enhanced transcription. Thus, H2A.B may participate in oncogenesis by promoting proliferation, which can be explained by its ability to enhance ribosome biogenesis. This is achieved by enhancing both rDNA and RP gene transcription. In Chapter 4, four novel PTMs on the N-terminus of H2A.B were discovered. These included R8me, R8me2a, S10ph, and R16me. Using a peptide pull-down approach, it was established that R8me2a interacted with the SWI/SNF complex and rRNA processing factors, while S10ph interacted with the Pol I transcription machinery. The unmodified H2A.B interacted with splicing factors and RPs. The authenticity of selected candidates as readers of H2A.B PTMs was confirmed. Chapter 5 shows that H2A.BR8me2a is both tightly chromatin-bound as well as soluble in nucleoplasm. The N-terminus of H2A.B is responsible for this dynamic exchange between this soluble and chromatin-bound pool. On the other hand, H2A.BS10ph was only observed to be tightly chromatin-bound. Interestingly, a splicing isoform of the Protein Arginine Methyltransferase 1 (PRMT1) was identified as the writer of the R8me2a modification. In Chapter 6, the functions of H2A.B in testes were shown to be conserved in HL, including enrichment at the TSS and gene body of active genes, positive correlation between H2A.B enrichment with the level of transcription, and the regulation of mRNA splicing. Also, for the first time, it was shown that H2A.B directly decondenses native chromatin in cells. This increase in chromatin accessibility directly increased the level of gene expression. Finally, a remarkable new function for H2A.B was uncovered; H2A.B regulates the genomic targeting of the chromatin remodelling complex SWI/SNF via its interaction with R8me2a. In summary, this thesis has uncovered a new mechanistic link between a testis-specific histone variant and cancer. My study has: (1) shown that H2A.B may play a role in HL oncogenesis by hyperactivating ribosomal biogenesis; (2) revealed a new aspect of the histone code hypothesis by showing that H2A.B is post-translationally modified. Significantly, the readers and writers of this H2A.B code were identified; (3) demonstrated that the epigenetic function of H2A.B in the testis, to regulate transcription and splicing, has been hijacked by HL and (4) uncovered a previous unknown connection between histone variants and ATP-dependent chromatin remodelling machines, two fundamental processes critical for the regulation of chromatin structure and function.