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

Area of Science:

• Stem Cell Biology
• Epigenetics
• Molecular Biology

Background:

• Generating induced pluripotent stem cells (iPSCs) from somatic cells using defined transcription factors (OCT4, KLF4, SOX2, C-MYC [OKSM]) is often slow and inefficient.
• Cellular barriers within differentiated cells resist changes in cell fate, hindering efficient reprogramming.
• Identifying molecular mechanisms that impede reprogramming is crucial for improving iPSC generation.

Purpose of the Study:

• To identify key molecular repressors that act as roadblocks during somatic cell reprogramming into iPSCs.
• To investigate the role of the identified repressors in the efficiency and speed of iPSC generation.
• To determine if targeting these repressors can enhance the generation of both mouse and human iPSCs.

Main Methods:

• An unbiased serial shRNA enrichment screen was employed to identify genes that suppress somatic cell reprogramming.
• The function of identified repressors, particularly SUMO2, was assessed by depleting them and evaluating reprogramming efficiency and kinetics.
• Experiments involved assessing transgene-independent, chimera-competent iPSC generation and evaluating the impact of SUMO2 suppression on human iPSC generation.

Main Results:

• The screen identified SUMO2, a protein modifier, as a potent repressor of iPSC formation.
• Depletion of SUMO2 significantly enhanced and accelerated the reprogramming process, yielding iPSCs in as little as 38 hours of OKSM expression.
• SUMO2 pathway inhibition promoted reprogramming independently of exogenous C-MYC and in parallel with small-molecule enhancers, and was effective in human cells.

Conclusions:

• The SUMO2 pathway represents a critical post-transcriptional barrier to acquiring pluripotency from fibroblasts using defined factors.
• Targeting sumoylation is a promising strategy to overcome somatic cell reprogramming barriers and improve iPSC generation efficiency and speed.
• Suppression of SUMO2 facilitates the generation of high-quality, transgene-independent iPSCs from both mouse and human cells.