Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

Abstract

Objective: To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediatorassociated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). Design: Experimental study. Setting: Academic research laboratory. Patient(s): Women undergoing hysterectomy for uterine fibroids. Intervention(s): Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. Main Outcome Measure(s): High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by in vitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. Result(s): A total of 296 unique phosphosites in 166 proteins were significantly decreased (R twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. Conclusion(s): These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

“…Alternatively, or additionally, Mediator kinase disruption could more directly impact replication dynamics, a possibility supported by recent studies that invoke a direct role for CDK8 and MED12, respectively, in replication origin firing and fork stability 45,51 . In this regard, among a catalog of high-confidence Mediator kinase substrates that we recently identified in myometrial stem cells, we note that transcription and replication associated proteins were prominently represented, consistent with either of these two possibilities 33 . Further studies will be required to determine the precise mechanism by which Mediator kinase disruption as a consequence of MED12 mutations triggers R-loop induced replication stress in UFs.…”

“…To this end, we treated UtSMCs with a highly specific chemical inhibitor of CDK8/19, CCT251545 32 , and monitored R-loop levels thereafter by immunocytochemical analysis using S9.6 antibody. Target engagement and Mediator kinase inhibition by CCT251545 under these experimental conditions was confirmed by analysis in parallel of INFγ-stimulated STAT1 phospho- www.nature.com/scientificreports/ rylation on serine residue 727 (pSTAT1 SER727 ), a validated biomarker of Mediator kinase activity in vivo 32,33 . This analysis revealed an inverse temporal relationship between Mediator kinase inhibition and R-loop formation.…”

“…CCT251545 in UtSMCs was performed by immunoblot analysis of STAT1 as described previously 33 using antibodies specific for total (SC-464; Santa Cruz Biotechnology) and phosphorylated (Ser727) (#9177; Cell Signaling Technology) STAT1.…”

Unveiling the noncanonical activation mechanism of CDKs: insights from recent structural studies

“…CDK8 also activates IFN-γ pathway by phosphorylating the STAT1 transcription factor (Steinparzer et al, 2019). Notably, a range of human CDK8/19 substrates, including DNA-binding transcription factors and chromatin associated proteins, has been identified in human cells, showing that the CDK8 plays diverse Frontiers in Molecular Biosciences frontiersin.org regulatory roles in various cellular processes (Poss et al, 2016;Barron et al, 2021).…”

Structural basis of the human transcriptional Mediator complex modulated by its dissociable Kinase module

“…In this regard, the cMED-CKM has been shown to interact with p-TEFb, which controls the release of paused Pol II 20 . Additionally, CDK8 phosphorylates several pausing-related factors, and inhibition of its kinase activity results in increased Pol II pausing 21–23 .…”