RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming

Abstract

Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

“…The three predominant isoforms of ZNF207 mRNA have been shown to differ by their inclusion of exons 6 and 9 (Fig. 3A) with exon 8/10 junctions exclusively present in isoform 2 [24]. Importantly, the three outliers were too short and lacked that isoform-defining junction (Fig.…”

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes

“…Using the STRING v10 database, the H3K9me3 heterochromatin proteins exhibited far more direct binding interactions among each other (Figure S4C, right) than with euchromatin proteins (Figure S4C, left) (Table S5). H3K9me3 heterochromatin proteins are enriched for repressors of iPS reprogramming over factors that enhance reprogramming, including 22 known iPS repressors (Bao et al, 2015; Han et al, 2013; Qin et al, 2014; Toh et al, 2016) (Figure 5E, S4D). …”

“…Of such proteins, TDP-43 ( TARDBP ), which forms neuronal inclusions in ALS (Taylor et al, 2016), has been shown to impede iPS reprogramming (Qin et al, 2014; Toh et al, 2016) (Figure S4D). Using transcriptome data from ALS patient fibroblasts (Highley et al, 2014), we find that germline mutations of TARDBP are associated with a widespread upregulation of genes in fibroblast srHC, compared to healthy controls (Figure S4E-G).…”

“… (E) Protein categories from proteomics analysis were compared to lists of iPS repressors (knockdown increases reprogramming) and iPS effectors (knockdown inhibits reprogramming) from a genome-wide screen (Toh et al, 2016). The ratio of repressors to effectors is plotted (numbers above the bar).…”

Mammalian synthetic biology in the age of genome editing and personalized medicine

“…Stockman et al developed a multiplex strategy (MoSAIC) for assessing genetic interactions using CRISPR/Cas9, in which direct barcoding and pairwise assembly of sgRNAs occur in a single cloning step, further improving assembly efficiency and reducing potential library bias associated with multi-step construction methods [37]. Pooled functional and phenotypic screens of RNAi- and CRISPRi-treated cells have revealed the relative impact of individual genes in mediating diverse cell behaviors, including somatic cell reprogramming, the unfolded protein response, and immune cell activation (Figure 2) [38–42]. CRISPR-mediated perturbations can also be carried out in vivo to analyze genetic determinants of physiological immune responses.…”