A Genome-Scale RNAi Screen for Oct4 Modulators Defines a Role of the Paf1 Complex for Embryonic Stem Cell Identity

Abstract

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

The transcriptional regulation of pluripotency

“…Indeed, large-scale RNAi knockdown studies have led to the discovery of important mESC factors such as Esrrb, Tbx3 and Tcl1 [26], as well as the chromatin regulators Tip60-p400 [27] and SetDB1 [28]. Similarly, unbiased genome-wide siRNA screens were able to identify Cnot3 and Trim28 [29], Paf1C [30] and the mediator and cohesin complex [31] as important mESC transcriptional cofactors. Extending this approach into hESCs, Chia et al [32] used a similar genome-wide siRNA screening to identify components of the INO80 chromatin remodeling complex, the mediator and TAF transcriptional regulatory complexes, and the COP9 signalosomes as important hESC factors.…”

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

“…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)(7)(8)(9)(10), tissue regeneration (11), viral infection (12), and antiviral response (13).…”

“…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 mouse ESCs (mESCs) for genes essential for the maintenance of ESC identity resulted in only ∼8% overlap (8,9), although many of the unique hits in each screen were known or later validated to be real. The lack of concordance suggest that these screens have not reached saturation (14) and that additional genes of importance remain to be discovered.…”

Cohesin and chromatin organisation

“…Several genome-wide RNAi screens have identified factors required for self-renewal, and cohesin and condensin have shown up time and time again [13,[63][64][65][66]. Since self-renewal implies proliferation, these screens will identify a large fraction of genes required for DNA replication, repair and cell division.…”