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

Abstract

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 G 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.

Comparative nuclear matrix proteome analysis of skeletal muscle cells in different cellular states

“…A large number of global studies at the level of the genome, transcriptome, and proteome have been carried out using these cells, revealing the muscle‐specific repertoire of gene expression profiles and regulatory networks and have served as a paradigm for understanding tissue‐specific gene regulation (Abdelmoez et al, 2020; Buckingham & Rigby, 2014; Forterre et al, 2014; Kislinger et al, 2005; Subramaniam et al, 2014; Tannu et al, 2004). Recent studies have also brought to light the role of epigenetic mechanisms in the regulation of skeletal muscle differentiation and regeneration (Barreiro & Tajbakhsh, 2017; Cheedipudi et al, 2015; Jin et al, 2016; Srivastava et al, 2018). However, the dynamics of the nuclear architecture in differentiating or quiescent muscle has not been adequately explored.…”

Is There a Histone Code for Cellular Quiescence?

“…Some of the gene expression, signaling, and functional changes observed with quiescence are likely specific for a cell type, while others are shared. Transcriptional changes with quiescence have been analyzed using cDNA libraries (Schneider et al, 1988;Coppock et al, 1993), microarrays (Venezia et al, 2004;Coller et al, 2006;Suh et al, 2012;Johnson et al, 2017), next generation sequencing (van Velthoven et al, 2017;Mitra et al, 2018b;Srivastava et al, 2018), and single-cell RNA sequencing methods (Kalakonda et al, 2008;Coller, 2019a). These studies demonstrated widespread gene expression changes with quiescence, some of which are functionally important for the quiescent state (Suh et al, 2012;Johnson et al, 2017;Lee H.N.…”