The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

The idea that liquid–liquid phase separation (LLPS) may be a general mechanism by which molecules in the complex cellular milieu may self-organize has generated much excitement and fervor in the cell biology community. While this concept is not new, its rise to preeminence has resulted in renewed interest in the mechanisms that shape and drive diverse cellular self-assembly processes from gene expression to cell division to stress responses. In vitro biochemical data have been instrumental in deriving some of the fundamental principles and molecular grammar by which biological molecules may phase separate, and the molecular basis of these interactions. Definitive evidence is lacking as to whether the same principles apply in the physiological environment inside living cells. In this Perspective, we analyze the evidence supporting phase separation in vivo across multiple cellular processes. We find that the evidence for in vivo LLPS is often phenomenological and inadequate to discriminate between phase separation and other possible mechanisms. Moreover, the causal relationship and functional consequences of LLPS in vivo are even more elusive. We underscore the importance of performing quantitative measurements on proteins in their endogenous state and physiological abundance, as well as make recommendations for experiments that may yield more conclusive results.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization.

TERT promoter mutations (TPMs) are the most common non-coding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual upregulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life-span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further upregulated to sustain cell proliferation.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5’ leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.