Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians

Abstract

E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation.

Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

“…Knockdown of several intestine-enriched transcription factors ( nkx2.2, gata4/5/6-1 ) causes reduced blastema formation and/or decreased neoblast proliferation (24, 37). Similarly, knockdown of the intestine-enriched HECT E3 ubiquitin ligase wwp1 causes disruption of intestinal integrity, reduced blastema formation, and neoblast loss (38). Conversely, knockdown of egfr-1 causes hyperproliferation and expansion of several neoblast subclasses (23).…”

Origin and evolution of fungal HECT ubiquitin ligases

“…A comparison between the sets of HECT genes in metazoa and fungi may provide additional insights on their early evolution. Figures 5 and 6 respectively show the maximum likelihood (ML) and neighbor-joining (NJ) trees corresponding to an alignment (Supplementary File 3 ) of the 203 sequences described above, plus a dataset already used in previous studies 20 , 23 , 36 , which includes HECT sequences of animals and the choanoflagellate Monosiga brevicollis as an outgroup. Both trees are largely congruent and show that all fungal genes have close animal relatives.…”

Ubiquitin-Dependent Regulation of Stem Cell Biology

“…In a similar manner, the E3 HUWE1, which has been linked to intellectual disability and schizophrenia, targets regulators of cell division, cell survival, and transcription, allowing it to promote the entry of neural precursor cells into the quiescent state [85–89]. Dependent on the tissue of origin of diverse stem cells, the multiple activities of the APC/C, SCF FBW7 , and HUWE1 likely endows these E3 ligases with the capacity to fine-tune the relationship between stem cell proliferation and quiescence programs, a function that for HUWE1 appears to be conserved all the way to planarians [90]. …”