An RNF12-USP26 amplification loop drives germ cell specification and is disrupted by disease-associated mutations

Abstract

The E3 ubiquitin ligase RNF12 plays essential roles during development, and the gene encoding it, RLIM , is mutated in the X-linked human developmental disorder Tonne-Kalscheuer syndrome (TOKAS). Substrates of RNF12 include transcriptional regulators such as the pluripotency-associated transcriptional repressor REX1. Using global quantitative proteomics in male mouse embryonic stem cells, we identified the deubiquitylase USP26 as a putative downstream target of RNF12 activity. RNF12 relieved REX1-mediated repression of Usp26 , leading to an increase in USP26 abundance and the formation of RNF12-USP26 complexes. Interaction with USP26 prevented RNF12 autoubiquitylation and proteasomal degradation, thereby establishing a transcriptional feed-forward loop that amplified RNF12-dependent derepression of REX1 targets. We showed that the RNF12-USP26 axis operated specifically in mouse testes and was required for the expression of gametogenesis genes and for germ cell differentiation in vitro. Furthermore, this RNF12-USP26 axis was disrupted by RLIM and USP26 variants found in TOKAS and infertility patients, respectively. This work reveals synergy within the ubiquitylation cycle that controls a key developmental process in gametogenesis and that is disrupted in human genetic disorders.

Cell autonomous requirement of imprinted XCI in extra-embryonic polar trophoblast cells

“…To investigate developmental roles of the Rlim-Rex1 axis in regulating iXCI, we first examined the expression profiles of both proteins during pre-implantation development. To obtain high quality antibodies, we developed a polyclonal Rex1 antibody raised in sheep recognizing aa 1-288 of mouse Rex1 (Segarra-Fas et al, 2022). Indeed, when tested on male embryonic stem cell (ESC) models by Western blot or by immunostaining, the Rex1 antiserum recognized Rex1 in nuclei of WT ESCs and ESCs carrying a Rlim deletion, but not ESCs carrying an additional Rex1 deletion (Suppl.…”