AbstractThe transcription factor WT1 is important in regulating physiological and pathological processes, including apoptosis, cell growth and cancer. BASP1 is a transcriptional co-suppressor of WT1; and together, these proteins constitute the WT1/BASP1 repressor complex. The WT1/BASP1 complex represses transcription by remodelling chromatin at the promoter region of WT1-target genes. Transcriptional repression by BASP1 requires its N-terminal myristoylation and a central role for lipids. Amongst other epigenetic modifications, the WT1/BASP1 complex deposits the repressive mark H3K27me3 on histone tails in the promoter region of WT1-target gene, yet the mechanism by which this occurs is undetermined. Previous experiments in the laboratory have indicated that EZH2 may play a role here. EZH2 is a core component of the polycomb repressor complex 2 (PRC2), alongside SUZ12 and EED. The PRC2 has been extensively linked to the deposition of H3K27me3 marks and inducing a transcriptionally repressive state at many target genes.
The current study used chromatin immunoprecipitation experiments to show a BASP1-dependent significant elevation in enrichment of EZH2 and SUZ12 at WT1-target gene promoters. Thus, this confirms that the intact PRC2 is recruited by WT1/BASP1 to WT1-target gene promoters. The recruitment of these two core PRC2 components was shown to be mediated independently of BASP1 myristoylation, suggesting that BASP1 directs transcriptional repression through both lipid-dependent and lipid-independent mechanisms.
Further experiments using RNA interference demonstrated a functionally relevant role for EZH2 in WT1-target gene repression. EZH2-targeted knockdown triggered an increase in AREG expression compared to the baseline level. The same dataset also suggested that both BASP1 and EZH2 work synergistically to invoke transcriptional repression, where no significant difference in AREG expression was observed during EZH2 knockdown compared to the combined knockdown of BASP1 and EZH2. Extended research integrating both RNA interference and ChIP techniques confirmed that trimethylation of H3K27 at WT1-target genes is EZH2-dependent, where knockdown of EZH2 led to a reduction in H3K27me3 enrichment at the AREG promoter.
Co-immunoprecipitation experiments failed to detect an interaction between BASP1 and the PRC2, and therefore it is proposed that the PRC2 is recruited to WT1-target genes following a BASP1-mediated alteration in the epigenetic landscape. Taken together, this study establishes a role for EZH2 and the intact PRC2 in BASP1-mediated deposition of H3K27me3 marks and BASP1-dependent transcriptional repression of WT1-target genes.
|Date of Award||25 Jan 2022|
|Supervisor||Karim T A Malik (Supervisor) & Stefan G E Roberts (Supervisor)|