Oct4 differentially regulates chromatin opening and enhancer transcription in pluripotent stem cells

The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD

A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05–15 mM, and a lower limit of urea detection was 20 μM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD

Application of butyrylcholinesterase‐based biosensor for simultaneous determination of different toxicants using inhibition and reactivation steps

Fast and simple detection of toxic compounds in aqueous solutions is important for agriculture and pollution monitoring. Enzyme‐based electrochemical biosensors are a promising platform for this task, but they usually lack the ability to distinguish between toxicants if more than one toxicant is present in the sample. Herein, we propose a new method of detection of various toxic compounds in complex multi‐component water samples using an electrochemical biosensor and additional stages of enzyme reactivation. The biosensor is based on butyrylcholinesterase immobilized on the surface of conductometric transducers using glutaraldehyde cross‐linking. It was shown that the biosensor is sensitive to organophosphorus pesticides, heavy metals ions, glycoalkaloids, but has a limited sensitivity to mycotoxins and surfactants. We propose a procedure for the analysis of complex samples with several reactivation stages to be able to determine which category of toxicants is present. Glycoalkaloids are reversible inhibitors and biosensor's activity is restored by washing in working buffer; heavy metal ions and pesticides are irreversible inhibitors and biosensor's activity is restored by incubation in EDTA and PAM‐2 solutions, correspondingly. This biosensor can be used for the detection of separate toxicants or for analysis of their mixtures in aqueous samples. It can be also used for the evaluation of total toxicity of the samples and applied for water quality monitoring

A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05-15 mM, and a lower limit of urea detection was 20 mu M. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis

Selective Mediator dependence of cell-type-specifying transcription

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks

Biological importance of OCT transcription factors in reprogramming and development

Ectopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences