Unravelling the Role of Bach1 and the CoREST Complex in Regulating Transcription

<p dir="ltr">Tight regulation of transcription is essential for cell function and the prevention of disease. This work has investigated both the mechanism of Bach1 transcriptional regulation, and assembly of the histone deacetylase (HDAC) complex, CoREST. Bach1 is a heme binding transcription factor. In a heterodimer with MafK, Bach1 represses genes involved in heme degradation. Upon heme binding, Bach1 dissociates from DNA and is thought to be degraded via the proteasome. The aim of this work was to further investigate this mechanism by which Bach1 regulates transcription, specifically when bound to heme. Utilising CRISPR/Cas9 gene editing, this work successfully established a tagged Bach1-FKBP12 HCT116 cell line, which allowed for rapid degradation of Bach1 upon 2h of dTAGv1 treatment. Western blotting then revealed in HCT116 cells, Bach1 is not completely degraded following hemin treatment. Furthermore, ChIP-seq revealed some residual Bach1 bound to DNA after hemin treatment. RNA-Seq analysis highlighted differences in transcription regulation following Bach1 degradation and hemin treatment. Additionally, pulldown and mass spectrometry results have suggested that Bach1 is able to recruit the Class I HDAC complexes to aid gene repression. This data therefore suggested that the mechanism by which Bach1 regulates gene expression is more complex than previously known, and thus an alternative non-degradation-based mechanism is proposed. HDACs are vital epigenetic enzymes which remove acetyl groups from histone tails, leading to gene repression. The bi-lobed structure of the CoREST complex was previously solved, but flexibility prevented higher resolution structures. Therefore, this thesis aimed to stabilise the CoREST complex by expressing additional binding partners. Utilising co-transfection, pulldowns, and mass-spectrometry, this work established that HMG20B does not directly interact with the complex. Unexpectedly, the CoREST complex purified with endogenous nucleosome. Purification was optimised and initial structural trials carried out.</p>