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.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene.

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs-myogenesis and the cell cycle-while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.

Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex.

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli are poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

Engineering Synthetic Signaling in Plants.

Synthetic signaling is a branch of synthetic biology that aims to understand native genetic regulatory mechanisms and to use these insights to engineer interventions and devices that achieve specified design parameters. Applying synthetic signaling approaches to plants offers the promise of mitigating the worst effects of climate change and providing a means to engineer crops for entirely novel environments, such as those in space travel. The ability to engineer new traits using synthetic signaling methods will require standardized libraries of biological parts and methods to assemble them; the decoupling of complex processes into simpler subsystems; and mathematical models that can accelerate the design-build-test-learn cycle. The field of plant synthetic signaling is relatively new, but it is poised for rapid advancement. Translation from the laboratory to the field is likely to be slowed, however, by the lack of constructive dialogue between researchers and other stakeholders.

Accelerating structure-function mapping using the ViVa webtool to mine natural variation.

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of these data remain inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family, or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well-studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less-studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project ( http://snp-seek.irri.org/) and human genetic variation ( https://www.ncbi.nlm.nih.gov/clinvar/).

Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis.

Regenerative potential in adult stem cells is closely associated with the establishment of-and exit from-a temporary state of quiescence. Emerging evidence not only provides a rationale for the link between lineage determination programs and cell cycle regulation but also highlights the understanding of quiescence as an actively maintained cellular program, encompassing networks and mechanisms beyond mitotic inactivity or metabolic restriction. Interrogating the quiescent genome and transcriptome using deep-sequencing technologies offers an unprecedented view of the global mechanisms governing this reversibly arrested cellular state and its importance for cell identity. While many efforts have identified and isolated pure target stem cell populations from a variety of adult tissues, there is a growing appreciation that their isolation from the stem cell niche in vivo leads to activation and loss of hallmarks of quiescence. Thus, in vitro models that recapitulate the dynamic reversibly arrested stem cell state in culture and lend themselves to comparison with the activated or differentiated state are useful templates for genome-wide analysis of the quiescence network.In this chapter, we describe the methods that can be adopted for whole genome epigenomic and transcriptomic analysis of cells derived from one such established culture model where mouse myoblasts are triggered to enter or exit quiescence as homogeneous populations. The ability to synchronize myoblasts in G0 permits insights into the genome in "deep quiescence." The culture methods for generating large populations of quiescent myoblasts in either 2D or 3D culture formats are described in detail in a previous chapter in this series (Arora et al. Methods Mol Biol 1556:283-302, 2017). Among the attractive features of this model are that genes isolated from quiescent myoblasts in culture mark satellite cells in vivo (Sachidanandan et al., J Cell Sci 115:2701-2712, 2002) providing a validation of its approximation of the molecular state of true stem cells. Here, we provide our working protocols for ChIP-seq and RNA-seq analysis, focusing on those experimental elements that require standardization for optimal analysis of chromatin and RNA from quiescent myoblasts, and permitting useful and revealing comparisons with proliferating myoblasts or differentiated myotubes.

Identification of PRDM2 regulated genes in quiescent C2C12 myoblasts.

Quiescent stem cells contribute to tissue homeostasis and repair in adult mammals. We identified a tumor suppressor PRDM2, as an epigenetic regulator induced in quiescent muscle stem cells as well as in cultured quiescent myoblasts. To delineate the functions of PRDM2 in muscle cells, we compared the gene expression profiles of control and PRDM2 knockdown myoblasts in growing, differentiating and quiescent conditions (GEO accession number: GSE 58676). To identify the direct targets of PRDM2 and the promoters co-associated with H3K9me2 (mark catalyzed by PRDM2), ChIP-Chip analysis was performed (GSE58748). In this report we discuss in detail the methodology used to identify PRDM2 regulated genes and classify them into potential direct and indirect targets.