A RNF12-USP26 amplification loop promotes germ cell specification and is disrupted in urogenital disorders

Abstract

SummaryUbiquitylation regulates all aspects of development, and components are frequently mutated in developmental disorders. Tonne-Kalscheuer Syndrome (TOKAS) is a X-linked multiple congenital anomaly disorder caused by mutations in the E3 ubiquitin ligase RNF12/RLIM and characterized by intellectual disability and urogenital abnormalities. However, the molecular underpinnings of TOKAS remain largely unknown. Here, we show that RNF12 catalytic activity relieves gene repression to drive a transcriptional program required for germ cell development and priming of pluripotent cells towards the germline. A major feature of the RNF12-dependent gametogenesis gene program is a transcriptional feed-forward loop featuring the deubiquitylase Usp26/USP26. Usp26/USP26 induction stabilises RNF12 to amplify transcriptional responses, which is disrupted by RNF12 TOKAS mutations and USP26 variants identified in patients with fertility defects. In summary, we uncover remarkable synergy within a ubiquitylation cycle that controls expression of key genes required for germ cell development and is disrupted in patients with urogenital abnormalities.

Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome

“…Immunoblotting analysis confirms that RNF12 RING domain mutations impaired substrate ubiquitylation in mESCs, as measured by accumulation of the REX1 substrate ( Fig 3D ). Furthermore, RNF12 RING domain mutations disrupted RNF12-dependent Usp26 gene expression, as measured by USP26 induction ( Fig 3D ), which we have previously shown is a transcriptional target of the RNF12-REX1 axis ( Segarra-Fas et al, 2020 Preprint ). Consistent with our in vitro data, UV-induced RNF12 crosslinking with photoABP was observed in mESC lysates expressing WT RNF12 ( Fig 3E ).…”

“…For this, we used CRISPR/Cas9 knock-in mESC lines harbouring RNF12 R575C (corresponding to human RNF12 R599C identified in TOKAS, see Fig 4A ) alongside RNF12 WT control or W576Y knock-in mESC lines ( Bustos et al, 2018 , 2020 ). RNF12 R575C and W576Y mutations result in REX1 accumulation ( Bustos et al, 2018 ; Segarra-Fas et al, 2020 Preprint ) and reduced expression of the RNF12-responsive gene Usp26 , again measured by USP26 protein levels ( Fig 4F ), confirming that these mutations impact on RNF12 activity. As expected, endogenous WT RNF12 was labelled by photoABP in these cell extracts.…”

“…RNF12 is expressed in a tissue-specific manner with enrichment in the brain ( Bustos et al, 2020 ) and is predominantly localised in the nucleus ( Jiao et al, 2013 ). RNF12 performs key developmental functions including regulation of gene dosage compensation via imprinted X-chromosome inactivation ( Gontan et al, 2012 , 2018 ) and regulation of developmental gene expression programmes ( Zhang et al, 2012 ; Bustos et al, 2018 , 2020 ; Segarra-Fas et al, 2020 Preprint ). RNF12 ubiquitylates several substrates, including REX1 ( Gontan et al, 2012 ), HDAC2 ( Krämer et al, 2003 ), CLIM1/2 ( Ostendorff et al, 2002 ), TRF1 ( Her & Chung, 2009 ), Stathmin ( Chen et al, 2014 ; Li et al, 2015 ), ERα ( Johnsen et al, 2009 ), c-Myc ( Gao et al, 2016 ), BRF1 ( Wang et al, 2019 ), and SMAD7 ( Zhang et al, 2012 ).…”

“…Interestingly, this region is also involved in allosteric regulation of catalysis ( Bustos et al, 2018 ). TOKAS variants in either region impair RNF12 substrate ubiquitylation ( Bustos et al, 2018 ; Frints et al, 2019 ), leading to disruption of developmental gene expression ( Bustos et al, 2020 ; Segarra-Fas et al, 2020 Preprint ).…”

Functions of SRPK, CLK and DYRK kinases in stem cells, development, and human developmental disorders

“… Germ cell differentiation [ 184 ] Phosphorylation of developmental E3 ubiquitin ligase RNF12/RLIM [ 93 , 178 ] Developmental angiogenesis in zebrafish [ 334 ] …”