Biology subjects

Camasses, A.

Publications and source records attributed to Camasses, A..

4 recordsLinked to original sources

bioRxiv2022

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also promotes transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell differentiation is unknown. Here, we find that CDK8 is dispensable for RNA polII CTD phosphorylation, regulation of gene expression, normal intestinal homeostasis and efficient tumourigenesis in mice. Furthermore, CDK8 is largely redundant with CDK19 in the control of gene expression. Yet, while their combined deletion in intestinal organoids reduces long-term proliferative capacity, it is not lethal and allows differentiation. Nevertheless, in double mutant organoids, the cystic fibrosis transmembrane conductance regulator (CFTR) pathway is transcriptionally and functionally downregulated, leading to mucus accumulation and increased secretion by goblet cells. This phenotype can be recapitulated by pharmacological inhibition of CDK8/19 kinase activity. Thus, the Mediator kinases are not essential for cell proliferation and differentiation, but they cooperate to regulate tissue-specific transcriptional programmes.

cell biology↗

bioRxiv2019

CDK8 and CDK19 kinases have non-redundant oncogenic functions in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common cancer with high mortality. The limited therapeutic options for advanced disease include treatment with Sorafenib, a multi-kinase inhibitor whose targets include the Mediator kinase CDK8. Since CDK8 has reported oncogenic activity in Wnt-dependent colorectal cancer, we investigated whether it is also involved in HCC. We find that CDK8 and its paralogue CDK19 are significantly overexpressed in HCC patients, where high levels correlate with poor prognosis. Liver-specific genetic deletion of CDK8 in mice is well supported and protects against chemical carcinogenesis. Deletion of either CDK8 or CDK19 in hepatic precursors had little effect on gene expression in exponential cell growth but prevented oncogene-induced transformation. This phenotype was reversed by concomitant deletion of TP53. These data support important and non-redundant roles for mediator kinases in liver carcinogenesis, where they genetically interact with the TP53 tumor suppressor.

cancer biology↗

bioRxiv2019

Mechanics of DNA Replication and Transcription Guide the Asymmetric Distribution of RNAPol2 and New Nucleosomes on Replicated Daughter Genomes

Eukaryotic DNA replication occurs in the context of chromatin. Chromatin in its capacity as a transcription regulator, is also thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is still unclear how chromatin structure survives the disruptions of nucleosomal architecture during genomic replication or if chromatin features are indeed instructive of the transcription state of the underlying gene. We have therefore developed a method for measuring chromatin structure dynamics after replication - ChIP -NChAP (Chromatin Immuno-Precipitation - Nascent Chromatin Avidin Pulldown) - which we used to monitor the distribution of RNAPol2 and new and old H3 histones on newly-replicated daughter genomes in S. Cerevisiae. The strand specificity of our sequencing libraries allowed us to uncover the inherently asymmetric distribution of RNAPol2, H3K56ac (a mark of new histones), and H3K4me3 and H3K36me3 ("active transcription marks" used as proxies for old histones) on daughter chromatids. We find a difference in the timing of lagging and leading strand replication on the order of minutes at a majority of yeast genes. Nucleosomes and RNAPol2 preferentially bind to either the leading or the lagging strand gene copy depending on which one replicated first and RNAPol2 then shifts to the sister copy after its synthesis has completed. Our results suggest that active transcription states are inherited simultaneously and independently of their underlying active chromatin states through the recycling of the transcription machinery and old histones, respectively. We find that "active" histone marks do not instruct the cell to reestablish the same active transcription state at its underlying genes. We propose that rather than being a consequence of chromatin state inheritance transcription actually contributes to the reestablishment of chromatin states on both replicated gene copies. Our findings are consistent with a two-step model of chromatin assembly and RNAPol2 binding to nascent DNA that is based on local differences in replication timing between the lagging and leading strand. The model describes how chromatin and transcription states are first restored on one and then the other replicated gene copy, thus ensuring that after division each cell will have "inherited" a gene copy with identical gene expression and chromatin states.

molecular biology↗

bioRxiv2017

Initiation of DNA replication requires actin dynamics and formin activity

Nuclear actin influences transcription in a manner dependent on its dynamics of polymerisation and nucleocytoplasmic translocation. Using human somatic cells and transcriptionally-silent Xenopus egg extracts, we show that actin dynamics is also required for DNA replication. We identify many actin regulators in replicating nuclei from Xenopus egg extracts, and show that in human cells, nuclear actin filaments form in early G1 and disassemble prior to S-phase. In either system, treatments that stabilise nuclear actin filaments abrogate nuclear transport and initiation of DNA replication. Mechanistically, actin directly binds RanGTP-importin complexes and disruption of its dynamics hinders cargo release. This prevents both nuclear pore complex (NPC) formation and active nuclear transport, which we show is required throughout DNA replication. Nuclear formin activity is required for two further steps: loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin and initiation of DNA replication. Thus, actin dynamics and formins are involved in several nuclear processes essential for cell proliferation.

cell biology↗