A Multiomics Approach to Elucidating the SpecificGenomic Targets Regulated by Histone Deacetylase 3(HDAC3)

Class I histone deacetylases (HDACs) form the catalytic core of 7 distinct corepressor complexes, capable of modulating gene expression, via the regulation of chromatin accessibility. HDAC3, unlike its HDAC1/2 counterparts, exclusively associates with the NCoR/SMRT corepressor complex, suggesting a unique function for the protein and the complex, which have not yet been elucidated. To investigate the biological relevance and distinguish the specific genomic functions of HDAC3 and the NCoR/SMRT corepressor complex, the CRISPR-Cas9-based dTAG system was leveraged. This enabled the rapid and inducible degradation of HDAC3 in HCT116 colon cancer cells on a timescale (>30 minutes) that had previously been unattainable. Using a combination of time-resolved multiomic techniques this study was able to comprehensively characterise the chromatin landscape, transcriptome, and proteome upon HDAC3 depletion, leading to the establishment of four new hypotheses for the regulation of gene expression by HDAC3 and the NCoR/SMRT complex. These included the coordinated repression with other HDAC complexes, association with transcription termination, RNA polymerase II pausing, and the deacetylation of non-histone targets. The study identified a core set of 12 genes regulated by HDAC3, encompassing negative cell cycle regulator p21 and long non-coding RNA, Keratins 8/18 and nuclear paraspeckle assembly transcript 1 (NEAT1). The transcriptomic analysis also revealed a previously unreported compensation mechanism between 6 and 24 hours for the loss of HDAC3 activity, before aberrant gene transcription prevailed, between 48 and 72 hours, accumulating in a multitude of proliferative and cell cycle defects. Overall, this study has provided a detailed insight into the multifaceted roles of HDAC3 exerts over the regulation of gene expression and cellular processes through the comprehensive analysis of multiomic datasets in HCT116 colon cancer cells.