A transcriptionally repressed quiescence program is associated with paused RNA polymerase II and is poised for cell cycle re-entry.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition. This article has an associated First Person interview with the first authors of the paper.

Building cross-border collaborations to increase diversity and accelerate rare disease drug development - meeting report from the inaugural IndoUSrare Annual Conference 2021.

The inaugural IndoUSrare Annual Conference was held virtually from 29 November to 2 December 2021 and was organized by the Indo US Organization for Rare Diseases (IndoUSrare). The event saw participation from over 250 stakeholders of rare diseases who joined in virtually by audio/video on the Zoom platform from around the world, with a majority of attendees concentrated in the Indian subcontinent and the United States. The conference was held over 4 days from 10:00 a.m. to 12:30 p.m. Eastern Time on each day, which accommodated participation by speakers and attendees from both the eastern and western hemispheres. The agenda over 4 days holistically covered broad topics of interest to different stakeholder groups such as representatives from organizations working toward policy frameworks for rare diseases or orphan drugs (Days 1, 4), biomedical research institutions (Day 2), patient advocacy organizations (Day 3), and patient advocacy and engagement offices within Industry (Day 4). In this meeting report, we summarize the key highlights from each day of this conference, with a perspective on future directions encouraging cross-border multistakeholder collaborations to maximize diversity, equity, and inclusion (DEI) in rare disease diagnosis, research, clinical trials, and treatment access. Each day included a keynote lecture on the theme of the day followed by a series of individual speaker presentations and/or a panel discussion. The goal was to understand current barriers and bottlenecks in the rare disease ecosystem. The discussions also helped highlight gaps and identify potential solutions that can be achieved through building multistakeholder collaborations across international borders, which we believe IndoUSrare is uniquely positioned to do with organizational programs such as rare patient foundation alliance, technology-enabled patient concierge, research corps, and corporate alliance program. The inaugural conference of the then 2+-year-old IndoUSrare organization laid the foundation for ongoing engagement of stakeholders between the two countries - the United States and India. The long-term goal is to scale the conference more broadly and serve as a model for other low- and middle-income countries (LMICs). Plain language summary: IndoUSrare held its inaugural Annual Conference from 29 November to 2 December 2021. It was focused on the theme of cross-border collaborations for rare disease drug development, with each day dedicated to a specific patient-focused discussion topic, ranging from patient-led advocacy (Advocacy Day), research (Research Day), rare disease community support and engagement (Patients Alliance Day), to industry collaborations (Industry Day). The 4-day conference was held in virtual mode and attracted over 250 attendees from across the globe. This meeting report provides the key highlights of the event and summarizes learnings and future directions encouraging cross-border collaborations to increase diversity, equity, and inclusion (DEI) in rare disease research and clinical trials.

A comparative analysis of important public clinical trial registries, and a proposal for an interim ideal one.

BACKGROUND: It is an ethical and scientific obligation to register each clinical trial, and report its results, accurately, comprehensively and on time. The WHO recognizes 17 public registries as Primary Registries, and has also introduced a set of minimal standards in the International Standards for Clinical Trial Registries (ISCTR) that primary registries need to implement. These standards are categorized into nine sections-Content, Quality and Validity, Accessibility, Unambiguous Identification, Technical Capacity, Administration and Governance, the Trial Registration Data Set (TRDS), Partner registries and Data Interchange Standards. This study compared the WHO's primary registries, and the US's ClinicalTrials.gov, to examine the implementation of ISCTR, with the aim of defining features of an interim ideal registry. METHODS AND FINDINGS: The websites of the 18 registries were evaluated for 14 features that map to one or more of the nine sections of ISCTR, and assigned scores for their variations of these features. The assessed features include the nature of the content; the number and nature of fields to conduct a search; data download formats; the nature of the audit trail; the health condition category; the documentation available on a registry website; etc. The registries received scores for their particular variation of a given feature based on a scoring rationale devised for each individual feature analysed. Overall, the registries received between 27% and 80% of the maximum score of 94. The results from our analysis were used to define a set of features of an interim ideal registry. CONCLUSIONS: To the best of our knowledge, this is the first study to quantify the widely divergent quality of the primary registries' compliance with the ISCTR. Even with this limited assessment, it is clear that some of the registries have much work to do, although even a few improvements would significantly improve them.

The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts.

BACKGROUND: Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood. RESULTS: Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro, but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1. CONCLUSIONS: Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

Mimicking Muscle Stem Cell Quiescence in Culture: Methods for Synchronization in Reversible Arrest.

Growing evidence supports the view that in adult stem cells, the defining stem cell features of potency and self-renewal are associated with the quiescent state. Thus, uncovering the molecular logic of this reversibly arrested state underlies not only a fundamental understanding of adult tissue dynamics but also hopes for therapeutic regeneration and rejuvenation of damaged or aging tissue. A key question concerns how adult stem cells use quiescence to establish or reinforce the property of self-renewal. Since self-renewal is largely studied by assays that measure proliferation, the concept of self-renewal programs imposed during non-proliferating conditions is counterintuitive. However, there is increasing evidence generated by deconstructing the quiescent state that highlights how programs characteristic of this particular cell cycle exit may enhance stem cell capabilities, through both cell-intrinsic and extrinsic programs.Toward this end, culture models that recapitulate key aspects of stem cell quiescence are useful for molecular analysis to explore attributes and regulation of the quiescent state. In this chapter, we review the different methods used to generate homogeneous populations of quiescent muscle cells, largely by manipulating culture conditions that feed into core signaling programs that regulate the cell cycle. We also provide detailed protocols developed or refined in our lab over the past two decades.