Ubiquitination is a barrier during reprogramming

Using a combination of gene ontology and gene network analysis we identified genes relevant to ubiquitination and the ubiquitin mediated proteolysis of pluripotency factors. In the canonical pathway ubiquitin is first activated (by E1 proteins), then conjugated (by E2 proteins), and then ligated (by E3 proteins) to target proteins; this ubiquitination often marks proteins for degradation. Ubuiquitin itself is liberated for reuse by de-ubiquitinating enzymes (DUBs) [1]. We identified multiple TRA-1-81+ screen hits in the ubiquitination pathway: an E2 conjugating enzyme UBE2D3 (p=0.0262), E3 ligases (FBXW7 p=0.004, NEDD4 p=0.0172, MARCH3 p=0.0152, RNF40 p=0.0044, RNF144a p=0.0002), and a DUB (USP9X p=0.0188). Thus, ubiquitination is targeted at multiple levels. Of note, ubiquitin ligase FBXW7 is significant in the TRA-1-81+ screen, as is its validated binding partner E2 enzyme UBE2D3. Fbxw7 knockdown has been shown to enhance reprogramming efficiency in mouse [2]. Similarly, in we identify the E2 conjugating enzyme UBE2D3 and its validated binding partner NEDD4 [3-4] as screen hits. Proteome array analysis shows NEDD4 to mediate ubiquitination of proteins responsible for such diverse processes as vesicle trafficking, extracellular matrix production, gene regulation, kinase activity, and protein binding events [5].