Regulation of H3K9me3 histone modifications during virus-specific T cell differentiation

CD8+ T cells or ¿cytotoxic T lymphocytes¿ (CTLs), serve a critical role in the elimination of virally infected and cancerous cells. Prior to antigen exposure, cytotoxic T cells exhibit a naïve or quiescent, phenotype and must undergo cellular differentiation upon activation to become effector cells enabling them to perform the functions required for clearance of infection or cancer. While it is understood that the changes in T cell phenotype and function that result from infection arise from specific changes in gene transcription, how these changes are regulated is not fully understood. It has previously been demonstrated from our lab that one means by which the changes in gene transcription are regulated during T cells differentiation is via chromatin remodelling (Russ et al., 2014). Our aim is to further analyse the chromatin changes by focusing on the repressive histone PTM H3K9me3, to understand its function in the complex regulatory process that results in the acquisition of effector cell functions and phenotype upon naïve CD8+ T cell activation. While it is known that H3K9me3 is regulated to control differentiation of various cell types, including CD4+ T cells (Allan et al., 2012), if and how H3K9me3 deposition regulates CTLs differentiation is unclear. In this thesis we showed that H3K9me3 deposition increases between naïve and effector CTLs. Further, infections of mice with strain A influenza virus (HKx31) in conjunction with chromatin immunoprecipitation and next generation sequencing (ChIP-seq) allowed us to map the genome wide distribution of H3K9me3 in naïve and effector CTLs. Comparison between the two subsets showed a major shift in the localization and regulation of H3K9me3 at key effector function gene promoters. This encouraged us to investigate further by comparing the mark distribution with chromatin structure and gene transcription levels to correlate changes in H3K9me3 on a global scale with changes in gene transcription and chromatin folding. The comparison of naïve CTL genome's map, constructed with high resolution Chromatin Conformation Capture (Hi-C) and genome-wide H3K9me3 distribution in the same cell subset showed a correlation between large H3K9me3 domains and topologically associated domains (TADs). The same comparison also helped pin-point cell specific enhancer-promoter interaction mediated by H3K9me3. In addition, RNA-seq analysis performed on naïve and recently activated CTLs indicated that the transcription level of a major portion of the genes controlled by these interactions quickly shifts upon TCR stimulation without following any significant pattern. These data all together suggest that H3K9me3 is more important in defining chromatin structure than in directly regulating gene transcription in the studied cell population. Overall this thesis provides an insight of the complex regulatory network that is behind T cell differentiation and of H3K9me3 contribution to it. Importantly, it shows a correlation between H3K9me3 and TADs structure. Finally, it proposes a novel molecular mechanism through which H3K9me3 can mediate cell specific enhancer-promoter interactions in within the TAD to pre-configure naïve CTLs to respond quickly upon TCR stimulation. Understanding how T cell differentiation is regulated is critical to enable immune interventions such as immunotherapy and novel augmented vaccination strategies.