Investigation of epigenetic cross-talk in the maintenance of genome integrity and gene expression

Abstract

Every cell in the human body carries the same genetic information, yet each performs a unique function, determined during its developmental pathway. This developmental diversity is orchestrated by complex epigenetic regulatory mechanisms. “Epigenetics” refers to heritable changes in phenotype that do not involve alterations in the DNA sequence. Epigenetic regulation is predominantly mediated by DNA methylation and histone modifications. Such modifications control the accessibility of chromatin to transcription factors, thereby regulating gene expression. In the cell nucleus, DNA is tightly packed with the help of octameric histone proteins forming a fundamental structure called chromatin. Histone proteins play essential role in both gene regulation and chromatin organization and in particular, covalent modifications in the N-terminal tail mediate these effects. Histone tails are subject to many different kinds of modifications such as acetylation, methylation and phosphorylation. Among the diverse histone modifications, histone lysine methylation, catalyzed by a group of enzymes called protein lysine methyltransferases (PKMTs) directly influence gene expression and chromatin states. Importantly, histone modifications rarely act in isolation; instead, they form combinatorial patterns known as the “histone code”, where each modification can influence others, establishing a cross-talk that drives specific gene regulatory outcomes. This thesis aims to elucidate the mechanisms of epigenetic cross-talk in maintaining genome integrity and gene expression. In the main project of this thesis, we systematically characterized the functional and molecular mechanism of the H3K9 tri-methyltransferase SETDB1. The triple tudor domain (3TD) of SETDB1 acts as a reader of H3K9 methylation in the presence of H3K14 acetylation facilitating the catalytic SET domain of SETDB1 to establish H3K9me3, thereby establishing silenced heterochromatin. This project focused on how the epigenetic cross-talk between H3K9me and H3K14ac modulates 3TD binding and H3K9 methylation by the SET domain of SETDB1. For this, a bottom-up approach was used to study the interaction between 3TD of SETDB1 and the histone H3 tail. A 3TD mutant with reduced H3-tail binding was generated and demonstrated the catalytic activity of SETDB1 on peptide containing H3K14ac and at the nucleosomal level harboring H3K14ac analog. Biochemical experiments demonstrated that H3K14ac is required for SETDB1 recruitment via 3TD binding to one H3 tail, facilitating H3K9 methylation on the other tail of the same nucleosome by the SETDB1 SET-domain. To validate these findings in a cellular system, a CRISPR-Cas9-mediated SETDB1 knockout model was generated and rescued with wild-type SETDB1, a 3TD-binding-deficient mutant (F332A), and a catalytically inactive mutant (H1224K). ChIP-seq data revealed a strong global correlation of H3K14 acetylation to catalytic activity and genomic binding of SETDB1. Furthermore, knockout of H3K14 acetyltransferase HBO1 was shown here to cause a drastic reduction in the H3K9me3 levels at SETDB1 dependent regions. Regions with hypomethylated DNA following SETDB1 loss also showed strong correlations with H3K9me3 and H3K14ac suggesting that lack of SETDB1 targeting also triggered loss of DNA methylation. Further genomic analysis indicated that the 3TD is particularly important for directing SETDB1 to L1M repeat elements. Together, these findings demonstrate that H3K14ac is necessary for SETDB1 recruitment and H3K9me3 deposition, revealing these marks as cooperative rather than antagonistic. The second project I was involved in, focused on the biological effects of DNA methylation at adenine residues (N6-methyl-adenine, m6dA) in mammalian cells. While m6dA is a well-characterized modification in prokaryotes, its occurrence and functional significance in mammals remains controversial due to its low abundance and the absence of known endogenous methyltransferases. To circumvent these technical peculiarities, an orthogonal approach to investigate the effects of m6dA in human DNA was adopted here by expressing well established bacterial methyltransferases CcrM to deposit m6dA in a sequence specific context. Subsequently, the cellular effects of this global site specific m6dA deposition were investigated. In this project, I contributed in evaluating the effect of m6dA in regulating PRC2 complex target genes. Gene set enrichment analysis from RNA-seq data revealed upregulation of PRC2 target genes following m6dA deposition. I validated this finding by performing H2K27me3 ChIP on selected significantly upregulated genes, demonstrating that catalytically active CcrM substantially reduced PRC2’s ability to deposit H3K27me3 at these regions, but not the inactive D39A mutant. These results highlight a functional interaction between m6dA and PRC2, suggesting that m6dA can regulate gene expression by interfering with PRC2 catalytic activity. This experimental model provides a foundation for further investigations into the role of m6dA in mammalian epigenetics. Altogether, this doctoral thesis offers insights into the cross talk of different epigenetic modifications forming an epigenetic code in maintaining genome integrity, chromatin states and the regulation of gene expression.