The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans

Abstract

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinasedependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals.KEYWORDS Mediator complex; CDK8; MED23; MED15; EGFR; Notch P RECISE regulation of transcription is required to execute developmental programs such as proliferation and cell fate determination. The Mediator complex ("Mediator") is a conserved eukaryotic transcriptional coregulator of RNA polymerase II (Pol II) transcription (Malik and Roeder 2010;Poss et al. 2013). Mediator consists of 30 subunits that assemble into four modules. "Core" Mediator consists of three of the four modules: the head and middle modules, which contact Pol II, and the tail module, which serves as a docking site for transcription factors. The fourth module, the dissociable cyclin dependent kinase 8 (CDK8) kinase module (CKM), interacts with transcription factors, core Mediator, chromatin, and the Pol II machinery to either repress or activate transcription (Malik and Roeder 2010;Nemet et al. 2014 transcription, tail and CKM subunits regulate specific transcriptional programs in animal development or physiology (Malik and Roeder 2010;Nemet et al. 2014). The CKM consists of enzymatic subunits CDK8 and cyclin C, and structural subunits MED12 and MED13 that tether the CKM to core Mediator (Tsai et al. 2013). C...

Integration of EGFR and LIN-12/Notch Signaling by LIN-1/Elk1, the Cdk8 Kinase Module, and SUR-2/Med23 in Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans

“…Thus, the LIN-39/Hox activator complex that promotes lateral signal gene transcription in P6.p may contain SUR-2 but not the CKM, and the LIN-1 repressor complex that prevents expression in other VPCs may not contain either SUR-2 or the CKM. We note that a small difference in penetrance of lag-2 expression reported for a complex background that differed in cdk-8 activity, interpreted as evidence that cdk-8 partially contributes to LIN-1-mediated repression of lateral signal genes (Grants et al 2016), is not statistically significant, so at present there is no unequivocal evidence for a partial contribution of the CKM to LIN-1-mediated repression of lateral signal genes.…”

“…When associated with the Mediator core complex, the CKM can sterically prevent RNA Pol II binding to cause transcriptional repression of target genes, or can promote transcriptional activation via the kinase activity of Cdk8. In C. elegans, the CKM has been implicated in the control of cell cycle quiescence of VPCs (Clayton et al 2008) and, when combined with mutations that activate EGFR pathway components or may have general effects on chromatin structure, in promoting ectopic vulval fate in VPCs that would normally adopt the 3°fate (Moghal and Sternberg 2003;Clayton et al 2008;Grants et al 2016). However, as discussed further herein, cdk-8 and cic-1/Cyclin C null mutants are homozygous viable and have overtly normal vulval development, suggesting that they are not required for normal VPC fate patterning; in contrast, dpy-22/Med12 and let-19/ Med13 null mutants are not viable, complicating the interpretation of their requirements in VPC fate patterning.…”

A broad mutational target explains an evolutionary trend

“…In C. elegans, the CDK-8 module acts in a highly pleiotropic fashion yet a P3.p division frequency phenotype was not previously reported. In the ventral epidermis, the CDK-8 module was shown to act at many other steps, contributing in the L1 stage to the fusion to hyp7 of anterior and posterior Pn.p cells (such as P2.p and P9.p) (Yoda et al, 2005), to the block of division of all VPCs in the L2 stage (Clayton et al, 2008) and to the level of induction of 2° and 1° VPC fates via cell-autonomous repression of EGF and Notch signalling in the L3 stage; these activities being mostly revealed in a sensitized genetic background (Moghal et al, 2003, Grants et al, 2016, Underwood et al, 2017. We found that mutation in three other genes encoding components of the CDK-8 module also increased P3.p division frequency in an otherwise wild-type genetic background ( Fig.…”

“…S13C,D): cic-1, dpy-22/mdt-12 and let-19/mdt-13. The valine-to-alanine substitution in the protein kinase domain found in MA line 450 likely causes a strong reduction-of-function, since the phenotypes such as dumpy animals or P3.p division frequency were indistinguishable from those in animals bearing the null deletion allele cdk-8(tm1238) (Grants et al, 2016) (Fig. S13C and Table S9).…”

cdc-25.4, aCaenorhabditis elegansOrtholog ofcdc25, Is Required for Male Mating Behavior

“…CDK-5 is also required for synapse elimination and formation (Park et al 2011), and for trafficking of glutamate receptors in the ventral nerve cord (Monteiro et al 2012). CDK-8 is required for axon navigation decisions in neurons (Steimel et al 2013), and also functions in vulval development by regulating the epidermal growth factor receptor-Ras-extracellular signal-regulated kinase pathway (Grants et al 2016). Taken together, these results suggest that CDKs phosphorylate not only cell cycle regulators, but also other regulators that are involved in diverse, important biological processes.…”