A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2

Abstract

SummaryThe generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.

SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States

“…Knockdown (KD) of Ubc9 led to a slight decrease in global sumoylation and clear appearance of free SUMO ( Figure S2A). Hyposumoylation increased iPSC formation (Figures 2A and 2B), a finding in line with the recent identification of Ubc9 and SUMO-2 as top hits in shRNA screens for enhanced reprogramming of fibroblasts to iPSCs (Borkent et al, 2016;Cheloufi et al, 2015). In Ubc9 KD cells, the level of the pluripotency factor Nanog was massively increased in comparison to control cells ( Figure 2C).…”

Heterochromatin and the molecular mechanisms of ‘parent-of-origin’ effects in animals

“…Recent RNAi screens have identified CAF-1, the SUMO-conjugating enzyme UBE2i, SUMO2, SETDB1, ATRX and DAXX as factors whose inhibition significantly enhances IPS reprogramming efficiency (Cheloufi et al 2015;Borkent et al 2016). All of these proteins are involved in regulation of heterochromatin (Cheloufi et al 2015;Borkent et al 2016) and heterochromatin-like domains (Figures 3 and 5; Table 1). RNAi ' knock-down' of CAF-1 also reduces levels of H3K9me3 at the RRRs (Cheloufi et al 2015) that are regions resistant to reprogramming at the 2-cell stage after SCNT (Matoba et al 2014).…”

Context-dependent requirement of H3K9 methyltransferase activity during cellular reprogramming to iPSCs

“…MEF cultures were established by trypsin digestion of midgestation (embryonic day (E) 13.5-E15.5) embryos and maintained in DMEM supplemented with 10% FBS, L-glutamine, penicillin/streptomycin, nonessential amino acids and β-mercaptoethanol. Reprogrammable MEFs were heterozygous for Rosa26-rtTA and for Oct4-GFP and either heterozygous for an inducible OKSM allele (Stadtfeld et al, 2010) or homozygous for an inducible OKS allele (Borkent et al, 2016). Established iPSCs were cultured on irradiated feeder cells in KO-DMEM (Invitrogen) supplemented with L-glutamine, penicillin/streptomycin, nonessential amino acids, β-mercaptoethanol, 1,000 U/ml LIF and 15% FBS ("ESC medium").…”