Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligandregulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. Steroid receptors are transcription factors regulated by physiological stimuli that dynamically bind to chromatin and control complex biological pathways (1). In particular, the glucocorticoid receptor (GR) is essential for life and is one of the most targeted proteins in the pharmacological industry due to its powerful antiinflammatory and immunosuppressive activities (2). Current pharmaceutical approaches are based on a recently challenged (3) binary model wherein direct binding of GR dimers and indirect binding of GR monomers via other proteins determine the transcriptional output (4).Upon hormone activation, GR associates to a subset of glucocorticoid response elements (GREs) across the genome, depending on the accessibility of the chromatin landscape (5). GR is a modular protein organized into three structural and functional domains; the N-terminal ligand-independent activation function-1 domain (NTD), the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) (6). GR, and all steroid receptors, are widely believed to bind DNA directly as homodimers (7). However, this paradigm has been established exclusively from in vitro studies, working mostly with the DBD fragment (8-10), only using the whole GR protein in rare cases (11,12). The small number of experiments performed in live cells only addresses the entire nuclear population, lacking specific information regarding the GR fraction bound to chromatin (13-16). Furthermore, these studies were unable to discriminate between dimers or higher oligomeric states.For the present study, we combine an experimental model where GR-DNA interaction can be observed in real time with techniques that allow the quantification of the oligomeric state of proteins inside living...

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Statins are potent inhibitors of HMG-CoA reductase, the key rate-limiting enzyme in cholesterol biosynthesis, and are some of the best selling drugs globally. We have explored the effect of chronic cholesterol depletion induced by mevastatin on the function of human serotonin(1A) receptors expressed in CHO cells. An advantage with statins is that cholesterol depletion is chronic which mimics physiological conditions. Our results show a significant reduction in the level of specific ligand binding and G-protein coupling to serotonin(1A) receptors upon chronic cholesterol depletion, although the membrane receptor level is not reduced at all. Interestingly, replenishment of mevastatin-treated cells with cholesterol resulted in the recovery of specific ligand binding and G-protein coupling. Treatment of cells expressing serotonin(1A) receptors with mevastatin led to a decrease in the diffusion coefficient and an increase in the mobile fraction of the receptor, as determined by fluorescence recovery after photobleaching measurements. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the organization and function of a G-protein-coupled neuronal receptor. Our results assume significance in view of recent reports highlighting the symptoms of anxiety and depression in humans upon statin administration, and the role of serotonin(1A) receptors in anxiety and depression.