A multi-omics integrative analysis based on CRISPR screens re-defines the pluripotency regulatory network in ESCs

Abstract

A comprehensive and precise definition of the pluripotency gene regulatory network (PGRN) is crucial for clarifying the regulatory mechanisms in embryonic stem cells (ESCs). Here, after a CRISPR/Cas9-based functional genomics screen and integrative analysis with other functional genomes, transcriptomes, proteomes and epigenome data, an expanded pluripotency-associated gene set is obtained, and a new PGRN with nine sub-classes is constructed. By integrating the DNA binding, epigenetic modification, chromatin conformation, and RNA expression profiles, the PGRN is resolved to six functionally independent transcriptional modules (CORE, MYC, PAF, PRC, PCGF and TBX). Spatiotemporal transcriptomics reveal activated CORE/MYC/PAF module activity and repressed PRC/PCGF/TBX module activity in both mouse ESCs (mESCs) and pluripotent cells of early embryos. Moreover, this module activity pattern is found to be shared by human ESCs (hESCs) and cancers. Thus, our results provide novel insights into elucidating the molecular basis of ESC pluripotency.

Stem cell transcriptional profiles from mouse subspecies revealcis-regulatory evolution at translation genes

“…The case for function of these regulators in stem cells is supported by a deep prior literature from laboratory mice. The top hit from our screen, Ctr9, is an integral member of the Pol II-associating factor 1 complex (Mueller and Jaehning 2002) with a well-characterized role in development and maintaining stem cell identity and pluripotency (Ding et al 2009;Ruan et al 2023). Direct binding by Ctr9 to translation genes in stem cells (Rahl et al 2010;Ding et al 2020) dovetails with our inference of Ctr9 regulation of this cohort, including its difference between lineages.…”

“…*, Fisher's exact test p = 0.02. (F) Data and symbols are as in Figure 1B except that each point reports a comparison of expression of one translation gene between Ctr9 knockdown and shRNA control mouse stem cells (Ruan et al 2023), and red and black text inlays report the percentage of translation genes where expression was higher in the indicated genotype with and without filtering for significance, respectively. S7.…”

“…Consistent with such a function, Ctr9 sites with sequence divergence between M. m. castaneus and its relatives coincided with divergent expression by the M. m. castaneus allele in hybrid stem cells, among translation genes (Figure 3E). Furthermore, our inference of Ctr9 as an activator for the translation pathway was borne out by expression profiles of Ctr9 knockdown in stem cells of laboratory mice (Ruan et al 2023), which established a marked directional influence of Ctr9 on translation genes (Figure 3F). Together, these data highlight Ctr9 as a case in which binding sites for a stem cell transcriptional regulator have evolved uniquely at translation genes in M. m. castaneus, representing candidate drivers of non-neutral expression divergence in this system.…”

“…For validation of the role of candidate transcription factors in translation regulation in stem cells, we analyzed published transcriptional data from embryonic stem cells with Ctr9 knocked down by shRNA (Ruan et al 2023; NCBI accession GSE219206, samples SRR22493339, SRR22493340, SRR22493341, and SRR22493342) and E8.5 embryos knocked out for Ehmt2 (Auclair et al 2016; NCBI accession GSE71500, samples SRR2133432, SRR2133433, SRR2133434, and SRR2133435), respectively.…”

“…For analysis of homozygous stem cells from (Skelly et al 2020) (genotypes C57BL/6J, A/J, 129S1/SvImJ, NZO/HILtJ, NOD/ShiLtJ, WSB/EiJ, CAST/EiJ, and PWD/PhJ), we downloaded raw reads from EBI's ArrayExpress database (Sarkans et al 2018) and mapped these to the respective reference genome. For validation of the role of candidate transcription factors in translation regulation in stem cells, we mapped RNA-seq from Ctr9 knockdown embryonic stem cells (NCBI accession GSE219206; Ruan et al 2023) and their corresponding shRNA control samples to the 129/SvImJ genome.…”