The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Cyclin-dependent kinase 9 (CDK9), the kinase component of positive transcription elongation factor b (P-TEFb), is essential for transcription of most protein-coding genes by RNA polymerase II (RNAPII). By releasing promoter-proximally paused RNAPII into gene bodies, CDK9 controls the entry of RNAPII into productive elongation and is, therefore, critical for efficient synthesis of full-length messenger (m)RNAs. In recent years, new players involved in P-TEFb-dependent processes have been identified and an important function of CDK9 in coordinating elongation with transcription initiation and termination has been unveiled. As the regulatory functions of CDK9 in gene expression continue to expand, a number of human pathologies, including cancers, have been associated with aberrant CDK9 activity, underscoring the need to properly regulate CDK9. Here, I provide an overview of CDK9 function and regulation, with an emphasis on CDK9 dysregulation in human diseases.

The concept of liquid-liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro. Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5-10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies.

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. On the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

We recently reported that the cyclin T1 histidine-rich domain creates a phase-separated environment to promote hyperphosphorylation of RNA polymerase II C-terminal domain and robust transcriptional elongation by P-TEFb. Here, we discuss this and several other recent discoveries to demonstrate that phase separation is important for controlling various aspects of transcription.

The post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes. Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.

BACKGROUND: Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins.
METHODS: This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes.
CONCLUSIONS: Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields.
CONCLUSIONS: Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. Although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Post-translational modifications of the carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAPII CTD) provide recognition marks to coordinate recruitment of numerous nuclear factors controlling transcription, cotranscriptional RNA processing, chromatin remodeling, and RNA export. Compared with the progress in yeast and mammals, deciphering the regulatory roles of position-specific combinatorial CTD modifications, the so-called CTD code, is still at an early stage in plants. In this review, we discuss some of the recent advances in understanding of the molecular mechanisms controlling the deposition and recognition of RNAPII CTD marks in plants during the transcriptional cycle and highlight some intriguing differences between regulatory components characterized in yeast, mammals, and plants.