Investigating the role of H3K4 demethylase enzymes in WT1-BASP1-mediated gene regulation

Abstract

The Wilms’ Tumour Protein 1 (WT1) is a transcriptional regulator that plays essential roles during development and in the maintenance of several adult tissues. WT1 can either activate or repress transcription, and the repressive function is mediated by interaction with the transcriptional co repressor Brain Acid Soluble Protein 1 (BASP1). To repress the transcription of WT1 target genes, BASP1 recruits chromatin remodelling enzymes. Previous work by our group has show that BASP1 recruits Histone Deacetylase 1 (HDAC1) to deacetylate the transcriptionally active histone H3K9ac mark. More recent studies have found that BASP1 is also responsible for the removal of H3K4me3 to mediate transcriptional repression. However, the histone demethylase responsible for demethylation at H3K4 has yet to be identified.

In this study, the relationship between BASP1 and the demethylase Lysine-Specific Demethylase 1 (LSD1), was assessed. Using chromatin immunoprecipitation (ChIP) it was demonstrated that BASP1 recruits LSD1 to the promoter region of WT1 target genes in K562 cells. Further ChIP studies showed that LSD1 is recruited by BASP1 when it is part of the coREST complex. Coimmunoprecipitation studies revealed that LSD1 and coREST also physically interact with BASP1, providing a mechanism for their co-recruitment to the promoter. The BASP1-mediated recruitment of LSD1 and coREST was disrupted by a mutation in BASP1 that perturbs the myristoylation site, suggesting that these events are dependent on the lipidation of BASP1. In contrast, similar analyses of histone H3K4me2 demethylation, the target of LSD1, does not require BASP1 myristoylation.

In order to assess a functional role for the BASP1-mediated recruitment of LSD1, RNA interference (RNAi) analyses were performed in MCF-7 breast cancer cells. Knockdown of either BASP1 or LSD1 resulted in an increase in the expression of WT1 target genes suggesting that they normally act to repress transcription of these genes. Knockdown of both BASP1 and LSD1 together resulted in an additive effect on transcription, indicating that they may work along separate pathways to induce transcriptional repression of WT1 target genes.

The results indicate that BASP1 interacts with and recruits LSD1 as part of the coREST complex to the promoter region of WT1 target genes. While LSD1 acts to repress transcription at WT1 target genes, it is not certain that this effect is dependent on BASP1. These findings expand upon previous work to identify and characterise the specific demethylase utilised by BASP1 for transcriptional repression.

Date of Award	12 May 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Karim T A Malik (Supervisor) & Stefan Roberts (Supervisor)