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.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

Exogenous chondroitin sulfate glycosaminoglycan associate with arginine-rich peptide-DNA complexes to alter their intracellular processing and gene delivery efficiency.

Arginine-rich peptides have been used extensively as efficient cellular transporters. However, gene delivery with such peptides requires development of strategies to improve their efficiency. We had earlier demonstrated that addition of small amounts of exogenous glycosaminoglycans (GAGs) like heparan sulfate or chondroitin sulfate to different arginine-rich peptide-DNA complexes (polyplexes) led to an increase in their gene delivery efficiency. This was possibly due to the formation of a 'GAG coat' on the polyplex surface through electrostatic interactions which improved their extracellular stability and subsequent cellular entry. In this report, we have attempted to elucidate the differences in intracellular processing of the chondroitin sulfate (CS)-coated polyplexes in comparison to the native polyplexes by using a combination of endocytic inhibitors and co-localization with endosomal markers in various cell lines. We observed that both the native and CS-coated polyplexes are internalized by multiple endocytic pathways although in some cell lines, the coated polyplexes are taken up primarily by caveolae mediated endocytosis. In addition, the CS-coat improves the endosomal escape of the polyplexes as compared to the native polyplexes. Interestingly, during these intracellular events, exogenous CS is retained with the polyplexes until their accumulation near the nucleus. Thus we show for the first time that exogenous GAGs in small amounts improve intracellular routing and nuclear accumulation of arginine-based polyplexes. Therefore, addition of exogenous GAGs is a promising strategy to enhance the transfection efficiency of cationic arginine-rich peptides in multiple cell types.

Non-metastatic 2 (NME2)-mediated suppression of lung cancer metastasis involves transcriptional regulation of key cell adhesion factor vinculin.

Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n=382), survival data (n=530) and lymph node metastases (n=100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2, did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i-mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells.

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.

NMR-based comparative metabolomics of quiescent muscle cells.

Adult muscle tissue largely comprised of differentiated myofibers also harbors quiescent muscle-resident stem cells (MuSCs) that are responsible for its maintenance, repair and regeneration. Emerging evidence suggests that quiescent MuSCs exhibit a specific metabolic state, which is regulated during physiological and pathological alterations. However, a detailed understanding of the metabolic state of quiescent MuSCs and its alteration during activation and repair is lacking. Direct profiling of MuSCs in vivo is challenging because the cells are rare and dispersed, while isolation and enrichment leads to their activation and loss of quiescence. In this study, we employed 1H-nuclear magnetic resonance (NMR) spectroscopy to profile metabolites in an established culture model of quiescent MuSC-derived myoblasts and compared with activated, proliferative and differentiated muscle cells to determine the state-specific metabolome. We report that the proliferating and differentiated cells are highly enriched in metabolites involved in energy generation, the quiescent state is enriched in metabolites related to phospholipid catabolism (glycerophosphocholine and choline) and depleted for phosphocholine which is enriched in proliferating cells. We propose that the ratio of these metabolites may be useful as a biomarker of MuSC quiescence.

Protocol for engineering and validating a synthetic mitochondrial intermembrane bridge in mammalian cells.

Membrane contact sites are recognized as critical means of intercompartmental communication. Here, we describe a protocol for engineering and validating a synthetic bridge between the inner and outer mitochondrial membranes to support functioning of the endogenous mitochondrial contact site and cristae organizing system (MICOS). A chimeric protein, MitoT, is stably expressed in cultured mammalian cells to bridge the mitochondrial membranes. This approach can be a valuable tool to study the function of the MICOS complex and associated proteins. For complete details on the use and execution of this protocol, please refer to Viana et al. (2021).

Adult Muscle Stem Cells: Exploring the Links Between Systemic and Cellular Metabolism.

Emerging evidence suggests that metabolites are important regulators of skeletal muscle stem cell (MuSC) function and fate. While highly proliferative in early life, MuSCs reside in adult skeletal muscle tissue in a quiescent and metabolically depressed state, but are critical for the homeostatic maintenance and regenerative response of the tissue to damage. It is well established that metabolic activity in MuSC changes with their functional activation, but the spatiotemporal links between physiological metabolism and stem cell metabolism require explicit delineation. The quiescent MuSC is defined by a specific metabolic state, which is controlled by intrinsic and extrinsic factors during physiological and pathological tissue dynamics. However, the extent of tissue and organismal level changes driven by alteration in metabolic state of quiescent MuSC is currently not well defined. In addition to their role as biosynthetic precursors and signaling molecules, metabolites are key regulators of epigenetic mechanisms. Emerging evidence points to metabolic control of epigenetic mechanisms in MuSC and their impact on muscle regenerative capacity. In this review, we explore the links between cell-intrinsic, tissue level, and systemic metabolic state in the context of MuSC metabolic state, quiescence, and tissue homeostasis to highlight unanswered questions.

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.