Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ER^T2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen—the active metabolites of tamoxifen—measured by LC-MS in these tissues. We also describe two cases of Cdk8^{floxed/floxed}/Rosa-Cre-ER^T2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Super-enhancers (genome elements that activate gene transcription) are DNA regions with an elevated concentration of transcriptional complexes. These multiprotein structures contain, among other components, the cyclin-dependent kinases 8 and 19. These and other transcriptional protein kinases are regarded as novel targets for pharmacological inhibition by antitumor drug candidates.

The precise balance of Th1, Th2, and Th17 cytokines is a key factor in successful pregnancy and normal embryonic development. However, to date, not all humoral factors that regulate and influence physiological pregnancy have been completely studied. Our data here pointed out cyclophilin A (CypA) as the adverse pro-inflammatory factor negatively affecting fetal development and associated with pregnancy complications. In different mouse models in vivo, we demonstrated dramatic embryotoxicity and teratogenicity of increased CypA levels during pregnancy. Using generated transgenic models, we showed that CypA overexpression in fetal tissues induced the death of all transgenic fetuses and complete miscarriage. Administration of recombinant human CypA in a high dose to pregnant females during fetal organogenesis (6.5–11.5 dpc) exhibited teratogenic effects, causing severe defects in the brain and bone development that could lead to malformations and postnatal behavioral and cognitive disorders in the offspring. Embryotoxic and teratogenic effects could be mediated by CypA-induced up-regulation of M1 macrophage polarization via activation of the STAT1/3 signaling pathways. Here, we propose secreted CypA as a novel marker of complicated pregnancy and a therapeutic target for the correction of pregnancy complications.

Blood malignancies often arise from undifferentiated hematopoietic stem cells or partially differentiated stem-like cells. A tight balance of multipotency and differentiation, cell division, and quiescence underlying normal hematopoiesis requires a special program governed by the transcriptional machinery. Acquisition of drug resistance by tumor cells also involves reprogramming of their transcriptional landscape. Limiting tumor cell plasticity by disabling reprogramming of the gene transcription is a promising strategy for improvement of treatment outcomes. Herein, we review the molecular mechanisms of action of transcription-targeted drugs in hematological malignancies (largely in leukemia) with particular respect to the results of clinical trials.

Transcriptional cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) are enzymatic components of the Mediator complex. CDK19 is presumably a minor paralog of CDK8 and is thought to compensate for lack of CDK8 in certain cases, but evidence for unique functions of the two kinases remains sporadic. Experiments with tumor cell lines have yielded the vast majority of information on the role and mechanisms of action of the kinases, and the results might be irrelevant because of the changes accumulated by tumor cells. Mice with an inducible Cdk8 knockout and a constitutive Cdk19 knockout ( Cdk 8 ^fl/fl / Cdk19 ^–/– / Rosa26/Cre/ER ^T2 ) were therefore engineered for the first time to study the joint role of CDK8 and CDK19 in the while organism and primary cell cultures. The mice were used to obtain Cdk19 ^–/– mouse embryonic fibroblasts (MEFs) in which a Cdk8 knockout could be induced with 4-hydroxytamoxifen (4-OHT). Unlike most tumor cells, MEFs were sensitive to CDK8/19 inhibition and knockout: the inhibition led to significant cell death within 5 days, while the knockout decreased the cell proliferation rate. RNA sequencing revealed alterations in the Wnt signaling pathway, cytokine response, and osteoclast differentiation in Cdk8/Cdk19 knockout MEFs. Expression of genes associated with steroidogenesis was lower, consistent with earlier data. Changes associated with the cytoskeleton, adipogenic differentiation, osteogenic differentiation, cell adhesion, extracellular matrix formation, and mitochondrial biogenesis were described for the first time. Studies of the stress responses in MEFs showed that responses to DNA damage from X-ray irradiation and to serum stimulation after starvation were also mediated by CDK8/19 and were significantly reduced in Cdk8/Cdk19 knockout cells.

CDK8 and CDK19 paralogs are regulatory kinases associated with the transcriptional Mediator complex. We have generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (iDKO) males, but not single Cdk8 or Cdk19 KO, had an atrophic reproductive system and were infertile. The iDKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single-cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star, and Fads) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes, and were likely associated with an impaired synthesis of steroids. Star and Fads were also downregulated in cultured Leydig cells after iDKO. The treatment of primary Leydig cell culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and a prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to the single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CCNC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CCNC stabilization.