| Popis: |
The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression. |
