We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.

The PBAF subtype of the mammalian chromatin remodeling SWI/SNF complex has wide and diverse functions in transcription regulation and development, being both transcription activator and repressor. ...

The PBAF chromatin-remodeling complexes are multi-protein machines, regulating expression of genes involved in proliferation and differentiation. PHF10 is a subunit of the PBAF essential for its association with chromatin. Mammalian PHF10 is expressed as four ubiquitous isoforms, which are alternatively incorporated in the complex and differ by their influence on transcription of target genes. PHF10 have different domain structure and two of them (PHF10-S isoforms) lack C-terminal PHD domains, which enables their phosphorylation by CK-1. Here we have found that PBAF subunits have low turnover rate, except for PHF10 which has much lower half-life, and is degraded by β-TrCP. The β-TrCP knockdown stabilizes PBAF core subunits - BRG1 and BAF155 and specific subunits - PHF10, BAF200, BAF180 and BRD7. PHF10 isoforms contain two non-canonical β-TrCP degrons and are degraded by β-TrCP in a phospho-dependent manner. But phosphorylation of PHF10-S degrons by CK-1, contrary to previously described degrons, prevents their degradation. Targeted molecular docking demonstrated that phosphorylated forms of PHF10 bind to β-TrCP with much lower affinity than non-phosphorylated ones, contrary to previously described degrons. This unorthodox mechanism proposes that phosphorylation of β-TrCP degrons by CK-1 could not only degrade a set of proteins, but also stabilize a different set of targets.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1–3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1–3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.