Functional analysis of RING and U-Box E3 ubiquitin ligases involved in stem cell regulation and regeneration in the planarian Schmidtea mediterranea

Regeneration is a widely distributed but not universal phenomenon in metazoans that involves the regrowth and repair of lost or damaged body parts that are damaged or lost. The dynamic process of regeneration requires the integration of wound response and patterning signals to establish a response that can regrow, repattern, and functionally integrate missing body parts. Ubiquitin is a small polypeptide with broad functions in cell biology including protein degradation, subcellular localization, and transcription. The specificity of ubiquitin signaling is controlled by the E3 ligases, a large protein family, that are understudied in the context of regeneration. The E3 ligases often act complexes including CRLs which utilize a cullin factor as an organizing scaffold and a substrate recognition factor, an example of which are the f-box genes as part of the SCF complex. We used the planarian, Schmidtea mediterranea, as a model organism to identify and investigate E3 ligases that regulate stem cells and regeneration. We used RNAi to perturb gene function for 103 RING/U-boxes, six cullins and 30 f-boxes and found phenotypes for 31 of these genes. We examined prpf19 and rnf2 in greater depth and for prpf19 found, using marker genes and TUNEL, that the basis of the phenotype was not stem cell loss as expected but rather a loss of progeny cells and an increase in apoptosis. rnf2 ubiquitylates H2A and functions within the epigenetic complex PRC1 to repress transcription. While rnf2(RNAi) demonstrated a mild phenotype, inhibition of PRC1 factor phc gave a striking phenotype of regional tissue misspecification. To understand the transcriptional targets of rnf2 and phc we used RNA-seq to understand and found surprisingly that phc and rnf2 largely regulated different target genes, explaining the differences in observed phenotypes. Using WISH we found striking spatial shifts in expression for phc target genes after phc(RNAi). These findings demonstrate key roles for E3 ligases in regeneration and stem cells and uncovered a role for cPRC1 in specifying regional tissue identity in planarians