Integrative framework for identification of key cell identity genes uncovers determinants of ES cell identity and homeostasis

Abstract

Identification of genes associated with specific biological phenotypes is a fundamental step toward understanding the molecular basis underlying development and pathogenesis. Although RNAibased high-throughput screens are routinely used for this task, false discovery and sensitivity remain a challenge. Here we describe a computational framework for systematic integration of published gene expression data to identify genes defining a phenotype of interest. We applied our approach to rank-order all genes based on their likelihood of determining ES cell (ESC) identity. RNAi-mediated loss-of-function experiments on top-ranked genes unearthed many novel determinants of ESC identity, thus validating the derived gene ranks to serve as a rich and valuable resource for those working to uncover novel ESC regulators. Underscoring the value of our gene ranks, functional studies of our top-hit Nucleolin (Ncl), abundant in stem and cancer cells, revealed Ncl's essential role in the maintenance of ESC homeostasis by shielding against differentiation-inducing redox imbalance-induced oxidative stress. Notably, we report a conceptually novel mechanism involving a Nucleolin-dependent Nanog-p53 bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our findings connect the dots on a previously unknown regulatory circuitry involving genes associated with traits in both ESCs and cancer and might have profound implications for understanding cell fate decisions in cancer stem cells. The proposed computational framework, by helping to prioritize and preselect candidate genes for tests using complex and expensive genetic screens, provides a powerful yet inexpensive means for identification of key cell identity genes.C ell identity is governed by a set of key regulators, which maintain the gene expression program characteristic of that cell state while restricting the induction of alternate programs that could lead to a new cell state. Identification of cell identity genes is a fundamental step toward understanding the mechanisms that underlie cellular homeostasis, differentiation, development, and pathogenesis. RNAi-based high-throughput screening has become a widely used method for identification of new components of diverse biological processes, including signal transduction, cancer, and host cell responses to infection (1, 2). Genome-scale RNAi screens have led to identification of tumor suppressors (3), oncogenes (4), therapeutic targets (5), and regulators of ES cell (ESC) maintenance (6-10), tissue regeneration (11), viral infection (12), and antiviral response (13).Despite the success of RNAi screens, false discovery and sensitivity remain a significant and difficult problem to address, with surprisingly small overlap among screen hits from independent but related screens (1). For example, multiple genomescale RNAi screens for host proteins required for HIV infection/ replication resulted in a limited overlap among screen hits at the gene level (1). Similarly, screens performed in mous...

Histone-Fold Domain Protein NF-Y Promotes Chromatin Accessibility for Cell Type-Specific Master Transcription Factors

“…ESCs lacking one or more of the NF-Y subunits exhibit severe self-renewal defects, undergo differentiation, and have global expression profile that is comparable to cells differentiating toward the trophoblast lineage, all consistent with the propensity of ESCs lacking many of these master ESC factors to differentiate towards trophoectoderm (Cinghu et al, 2014; Freudenberg et al, 2012; Li et al, 2007; Nichols et al, 1998). In light of overexpression of NF-YA counteracting ESC differentiation induced by Leukemia inhibitory factor (LIF) withdrawal (Dolfini et al, 2012b), NF-Y’s broad control of key ESC identity genes including master ESC TFs places NF-Y as part of the core pluripotency network.…”

“…This assumption may have unintentionally led to the underappreciation of the roles “housekeeping” or other essential TFs, expressed ubiquitously or in a multitude of cell types, might play in cell specification. Recent studies, however, have shown that many ubiquitous factors, previously thought to have an exclusive housekeeping function, have additional cell type-specific roles (Chia et al, 2010; Cinghu et al, 2014; Golan-Mashiach et al, 2012; Kagey et al, 2010; Kim et al, 2010a; Pijnappel et al, 2013). This emerging body of evidence indicates that we are yet to fully appreciate the significance of ubiquitously expressed proteins in many settings, and argues for a comprehensive reassessment of the roles other housekeeping proteins may play in cell specification.…”

An Apela RNA-Containing Negative Feedback Loop Regulates p53-Mediated Apoptosis in Embryonic Stem Cells

“…Nucleolin and aurora kinase A (Aurka) inhibit the pro-differentiation activity of p53 in mESCs (Cinghu et al, 2014; Lee et al, 2012; Montes de Oca Luna et al, 1995). However, it is unknown which factor(s) suppresses the pro-apoptotic function of p53 in mESCs.…”

Intragenic Enhancers Attenuate Host Gene Expression

“…Mouse ESCs (E14Tg2a, ATCC) were maintained on gelatin-coated plates in the ESGRO complete plus clonal grade medium (Millipore), as previously described (Cinghu et al, 2014; Oldfield et al, 2014). For experiments, ESCs were cultured on gelatin-coated plates in M15 medium: DMEM (Invitrogen) supplemented with 15% FBS, 10μM 2-mercaptoethanol, 0.1 mM nonessential amino acids (Invitrogen), 1× EmbryoMax nucleosides (Millipore), and 1U/ml of ESGRO mLIF (Millipore).…”

“…Intragenic enhancer-containing genes in ESCs were analyzed in relation to the rank-ordered list of all mouse genes (Cinghu et al, 2014) that are likely to be associated with ESC maintenance vs. ESC differentiation.…”