Multi-omics Investigation of the MiDAC Deacetylase Complex

Histone deacetylases (HDACs) are enzymes responsible for removing of acetyl groups from histone tail lysine residues, which cause changes in gene transcription. HDAC transcriptional regulation is important in cellular and developmental functions including cell cycle, proliferation and embryogenesis. Mutations and upregulation of these proteins has been linked to various diseases such as Alzheimer’s, Parkinson’s and various cancers. HDAC inhibitors have been used to treat diseases, however currently available inhibitors lack specificity. Greater understanding of HDAC structure and function could improve targeting and so is an important area of investigation.

Mitotic deacetylase complex MiDAC is one of seven multiprotein complexes which are formed by Class I HDACs (1, 2 and 3). It is the most recently discovered complex, containing MIDEAS and DNTTIP1 proteins. It has been shown that the homozygous MIDEAS or DNTTIP1 knockout mouse embryos die before embryonic day 16.5 with perturbed haematopoiesis and heart malformations. The MiDAC complex has also been shown to regulate neuron outgrowth and affect neuronal development. Although MiDAC clearly plays an essential role, the complex and its primary gene targets are not well characterized.

The experiments in this thesis involve tagging of endogenous MIDEAS and DNTTIP1 genes with an FKBP12F36V/3xFLAG tag in HCT116 cells using CRISPR-Cas9 approach. The FKBP12F36V proteolysis tag in combination with the dTAGv1 proteolysis targeting chimera allows rapid and complete protein depletion in under an hour. Following rapid depletion of tagged DNTTIP1 and MIDEAS, histone blots revealed increase in acetylation of several histone marks. ATAC-seq experiment suggested that MiDAC targets only a few specific genes in the genome. RNA-seq and qPRO-seq approaches revealed early gene changes, which may be considered primary transcriptional targets with possible links to reported phenotypes. The results suggest that MiDAC may have a unique and specialized function, crucial in development and embryogenesis.