CDK8/19 in stress response using mouse embryonic fibroblasts model

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Resumo

Transcriptional cyclin-dependent kinases CDK8 and CDK19 are enzymatic components of the Mediator complex. CDK19 is supposed to be a minor paralog of CDK8, but, probably, in some situations it can compensate for the absence of CDK8, while evidence for unique functions of both kinases remains sporadic. The vast majority of information on the role and mechanisms of action of these proteins was obtained in experiments using tumor cell lines, which may give irrelevant results due to the changes accumulated by tumor cells. In this regard, we first obtained mice (Cdk8fl/fl/Cdk19–/–/Rosa26/Cre/ERT2) with inducible Cdk8 knockout on the background of constitutive Cdk19 knockout to study their joint role in the whole organism and in primary cells. Using these mice, we obtained Cdk19–/– embryonic fibroblasts, with Cdk8 knockout inducible by 4-hydroxytamoxifen. We found that, unlike most tumor cells, embryonic fibroblasts are sensitive to CDK8/19 inhibition and knockout: inhibition resulted in a significant cell death already at day 5, while knockout resulted in a decrease in their proliferation rate. RNA sequencing revealed, consistent with previously published studies, alterations in the Wnt signaling pathway, cytokine response, and osteoclast differentiation. Consistent with our previously published results, expression of genes associated with steroidogenesis was reduced. Changes associated with the cytoskeleton, adipogenic differentiation, osteogenic differentiation, cell adhesion, extracellular matrix formation, and mitochondrial biogenesis were previously undescribed. When studying the stress response of embryonic fibroblasts, we found that the response to DNA damage from X-ray irradiation and to serum stimulation after starvation was also mediated by CDK8/19 and was significantly reduced in knockout cells.

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Sobre autores

E. Varlamova

Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Autor responsável pela correspondência
Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 119334

T. Kirukhina

Institute of Gene Biology, Russian Academy of Sciences

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334

A. Isagulieva

Institute of Gene Biology, Russian Academy of Sciences; Burnazyan Federal Medical Biophysical Center, Federal Medical Biological Agency of the Russian Federation

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 123098

A. Khamidullina

Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 119334

M. Sorokina

Almazov National Medical Research Centre, Ministry of Health of the Russian Federation

Email: katerinavarlamova196@gmail.com
Rússia, St. Petersburg, 197341

Yu. Silaeva

Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 119334

V. Tatarskiy

Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 119334

A. Bruter

Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: katerinavarlamova196@gmail.com
Rússia, Moscow, 119334; Moscow, 119334

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2. Fig. 1. CDK8/19 are required for the viability of mouse embryonic fibroblasts (MEFs) in vitro. a — Confirmation of CDK8 elimination in MEFs after 72 h of incubation in the presence of 4-hydroxytamoxifen (4-OHT). Each lane represents a MEF line derived from a single embryo. b — Cyclin C levels are reduced to undetectable levels only in the absence of both CDK8 and CDK19 kinases. c — Fourteen days after induction of Cdk8/Cdk19 gene knockout (iR/8–/19–), the proliferative activity of MEFs is reduced by 1.5-fold compared to wild-type (WT) MEFs. g — Distribution of MEFs with Cdk8/Cdk19– gene knockout (iR/8–/19–) by cell cycle phases does not differ from the distribution of control MEFs without induction (R/8fl/19–) throughout the entire experiment (21 days). G0-G1 corresponds to the cell cycle phase with a diploid chromosome set; G2-M — with a tetraploid chromosome set; S — synthetic phase of the cycle; subG1 fraction — cells with fragmented DNA (dead cells). d — Senexin B causes dose-dependent cell death of wild-type MEF cells within 5 days (IC50 = 0.39 ± 0.7 μM). e — Senexin B at a concentration of 1 μM leads to the death of 72.0 ± 6.9% of wild-type MEF cells. The level of pRb phosphorylation at serine residues 807/811 (phospho-pRb S807/811) is reduced 14 days after Cdk8 knockout induction in MEF iR/8–/19– cells (g) and after 72 h of incubation in the presence of 1 μM senexin B (h). Densitometric analysis of Western blots was performed in three (g) and six (h) biological replicates for MEF cells obtained from different embryos (ns – data are statistically insignificant, *p < 0.05).

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3. Fig. 2. In mouse embryonic fibroblasts (MEFs), processes associated with inflammation response, metabolism, and cytoskeletal dynamics change 14 days after induction of Cdk8/Cdk19 knockout. a — Dot plot of the dependence of differential gene expression (DEG) on the lgp_adjust value. Genes with insignificant changes (p_adjust < 0.05) are shown in gray. Genes with functions described in more detail in the text are shown in green. Results of enrichment of biological processes (GO analysis) (b) and molecular pathways (according to the KEGG database) of DEGs (c). Designations: WT — R/8fl MEF without induction (wild-type fibroblasts), iR/8–/19– — MEF with Cdk8/Cdk19 knockout. The analysis was performed in R using the clusterProfiler library. g — In iR/8–/19– MEF, the regulation of expression of genes encoded by the mitochondrial genome (mt-Nd4, mt-Nd5, mt-Nd2, mt-Nd6, mt-Te, mt-Rnr, mt-Cytb) and nuclear genes encoding mitochondrial proteins (Lars2 and Trmt112-ps2) is disrupted. d — In iR/8–/19– MEF, expression of various non-coding RNAs is altered.

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4. Fig. 3. CDK8/19 deficiency results in decreased levels of key proteins involved in various cellular processes. Western blot analysis of MEFs 3 and 14 days after Cdk8 gene knockout induction with 1 μM 4-hydroxytamoxifen (4-OHT). The decreased levels of β-catenin, p65, and ERK1/2 proteins support the RNA sequencing results of iR/8–/19– MEFs.

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5. Fig. 4. CDK8/19 are involved in the MEF response to serum stimulation and DNA damage. a — MEFs proliferate more slowly in response to stimulation with 10% DMEM in the absence of CDK8/19. The change in proliferation rate is calculated relative to the first day of stimulation. b — Dynamics of changes in cell distribution by cell cycle phases for 24 h after addition of 10% DMEM. c — Knockout of the Cdk8/Cdk19 genes, in contrast to inhibition of their kinase activity, leads to less pronounced induction of serum response genes. ns — changes are statistically insignificant, *p < 0.05, ****p < 0.00001. d — Results of Western blotting of total protein of wild-type and iR/8–/19– MEFs 48 and 74 h after exposure to X-ray irradiation at a dose of 4 or 7 g. Cdk8/Cdk19 knockout MEFs exhibit a less pronounced DNA damage response and higher levels of apoptosis activation marker (p53).

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