The interplay between Heterochromatin Protein (HP1) and histone variant H2A.Z in gene expression regulation
Creators: name: Azad, Bhumika
Publisher: name: The Australian National University
Year: 2019

description: It is now clearly established that the central regulator of eukaryotic gene transcription is the organization of the genome into chromatin. Chromatin performs this crucial function by partitioning the genome into functionally specialised domains and subdomains, that is heterochromatin and euchromatin. Chromatin is built from nucleosomes, the universal repeating protein-DNA complex in all eukaryotic cells. Underpinning the proper regulation of gene expression are epigenetic-based mechanisms that regulate the structure of chromatin. These include histone modifications, the incorporation of histone variants and the binding of architectural chromatin proteins (ACPs). ACPs are universal nuclear proteins that utilise the structural properties of chromatin to create functionally unique higher-order chromatin structures. The most intensively studied ACP is HP1. HP1 is a highly conserved and essential silencing protein. Mammalian HP1 exists in three isoforms; HP1a, HP1b, and HP1g. It is still unknown why these three different isoforms exist? In thesis, I examined whether HP1a and HP1b have unique or overlapping functions in determining the phenotype of a cell and in regulating gene expression. The different HP1 isoforms have received considerable attention recently because they are aberrantly expressed in cancers. However, their role is controversial because in some cancers the expression of certain isoforms increases and in other types of cancers, it decreases. Further, how they contribute to cancer is unclear. In this thesis, I investigated whether altering the expression of HP1a or HP1b is sufficient to drive the major phenotypic changes associated with malignancy and also examined the associated changes in gene expression. Chromatin is a complex structure comprised of a multitude of different biochemical components. Therefore, no individual component of chromatin works in isolation and its function can be modulated by other components of chromatin. One important way the structure and function of chromatin is regulated is by the replacement of the major histones with their variant forms. Significant to the function of HP1 is the essential histone variant H2A.Z. Results from the Tremethick laboratory have revealed that HP1a is a reader of H2A.Z and moreover, they cooperate to form compacted chromatin structures. Finally, I investigated the interplay between H2A.Z and HP1 on regulating gene expression and changes to cellular morphology and function. To address these aims, isogenic MCF10A (non-invasive) and MCF10Ca1a (invasive) human breast epithelial cell lines were employed. Specifically, I established single and double MCF10A knockdown cell lines and compared the associated phenotypic changes with the malignant MCF10Ca1a cell line. In addition, I performed RNA and ChIP-seq experiments to study the impact of these knockdowns of HP1 and/or H2A.Z on gene expression. The major conclusions of this thesis: 1) the knockdown of HP1a and HP1b had different effects on cellular proliferation and cell migration indicating that they have different cellular functions; 2) neither the single nor double knockdown of HP1a and HP1b induced a MCF10Ca1a malignant-like phenotype. However, the combined knockdown produced an interesting cellular change, in which these shHP1a and shHP1b cells stopped dividing and entered the G0-G1 stage of the cell cycle. 3) most interestingly, the combined depletion of HP1a and HP1b showed that a large number of misregulated genes were involved in important centromere organisation and functions; 4) while RNA-seq data indicated that loss of HP1a can be compensated by HP1b and vice versa, unexpectedly the ChIP-seq data revealed that this was not the case suggesting that proper gene regulation can be maintained by sub optimal amounts of HP1a or HP1b; and 6) H2A.Z was required for the recruitment of HP1a and HP1b on the gene bodies of inactive and active genes, as well as on centromeric DNA repeat elements.