Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions.

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells show hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi-enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.