Functional studies of genome architectural proteins CTCF and ZNF143

Abstract

The genome architecture and gene transcription are interdependent. The way chromatin is packed within the nucleus controls when and how genes are expressed. A lot of chromatin interacting proteins are involved to ensure regulated gene transcription. The highly conserved zinc finger protein CCCTC-binding factor (CTCF) is one of the core genome architecture proteins that acts as a transcription insulator and activator where it regulates long-range chromatin and contributes to the establishment of topological-associating domains (TAD). Genome-wide analyses suggested that zinc finger 143 (ZNF143), another ubiquitously expressed zinc transcription factor is also a key regulator for CTCF-mediated chromatin interactions to establish conserved chromatin loops. In addition, chromatin remodelling protein chromodomain helicase DNA binding protein 8 (CHD8) has also been shown to colocalise extensively at CTCF and ZNF143 binding sites to modulate nucleosome positions near chromatin loop anchors. Recent studies are increasingly describing the importance of the crosstalk between these chromatin proteins and how they play a significant role in the three-dimensional genome architecture, however a lot of the underlying mechanisms of chromatin organisation remain unclear. This thesis focuses on the functional roles of three chromatin proteins, CTCF, ZNF143 and CHD8, to gain an insight into how these proteins might interact with each other and probe their role in chromatin organisation. Purification protocols of human CTCF, ZNF143 and CHD8 were established. Detailed functional assays were carried out on the individual proteins to provide insights on how they might act on the genome. Their DNA- and nucleosome-binding properties were investigated with biochemical and biophysical techniques. Direct interactions between these proteins were further explored with pull-down assays. Structural analysis was performed on the CTCF-CHD8 complex using negative stain and cryo-electron microscopy. While CHD8 was able to interact with both CTCF and ZNF143, direct interaction between CTCF and ZNF143 was not observed, indicating that CHD8 might be a cofactor that mediates the association of CTCF and ZNF143 at chromatin loop anchors. Together, the biochemical and biophysical studies presented in this thesis provide a fitting contribution to our current knowledge about the molecular mechanisms in genome organisation.