Abstract
Glis2/NPHP7 is a transcriptional regulator mutated in type 7 nephronophthisis, an autosomal recessive ciliopathy associated with cystic and fibrotic kidney disease as well as characteristic extrarenal manifestations. While most ciliopathy-associated molecules are found in the cilium, Glis2/NPHP7 presumably localizes to the nucleus. However, the detection of endogenous Glis2/NPHP7 has remained unsuccessful, potentially due to its ubiquitylation-dependent rapid degradation. We report now that Glis2/NPHP7 is also SUMOylated, preferentially by PIAS4, which conjugates Glis2/NPHP7 to SUMO3. SUMOylation interferes with ubiquitylation and degradation of Glis2/NPHP7, suggesting that Glis2/NPHP7 protein levels are regulated by competing ubiquitylation/ SUMOylation. SUMOylation also alters the transcriptional activity of Glis2/NPHP7. While Glis2/NPHP7 activates the mouse insulin-2-promotor (mIns2), SUMOylated Glis2/NPHP7 lacks this property, and seems to act as a repressor. Taken together, our data reveal that Glis2/NPHP7 is extensively modified by post-translational modifications, suggesting that a tight control of this transcriptional regulator is required for normal development and tissue homeostasis.
Highlights
Glis2/NPHP7 is one of three proteins that constitute the family of Glis-similar proteins (Glis13), a sub-family of Krüppel-like transcription factors characterized by five tandem Cys2/His2 zinc fingers
Since TRIM32 interacts with the E3 SUMO ligase PIAS4/PIASγ [17] and SUMOylation often targets functionally or physically connected proteins [18], we examined whether Glis2 directly or indirectly interacts with PIAS4 and SUMO machinery
The direct physical interaction between PIAS4 and Glis2 was further confirmed by co-immunoprecipitation, using proteins produced by cell-free wheat germ system (S1A Fig).Co-localisation of fluorescently tagged Glis2 and PIAS4 in the nucleus further supported a functional interaction between these two proteins (Fig 1B)
Summary
Glis2/NPHP7 is one of three proteins that constitute the family of Glis-similar proteins (Glis13), a sub-family of Krüppel-like transcription factors characterized by five tandem Cys2/His zinc fingers (reviewed in [1]). While all three Glis proteins are expressed in the kidney, Glis2/NPHP7 mRNA is detectable in several extra-renal tissues [2,3]. Consistent with their function as transcriptional regulators, Glis proteins localize predominantly to the nucleus, requiring the PLOS ONE | DOI:10.1371/journal.pone.0130275. SUMOylation Blocks Ubiquitylation of Glis/NPHP7 integrity of the zinc finger domains [4]. While Glis represses the Gli1-mediated activation of a reporter construct containing Glis-binding sequences, Glis activates the mouse Insulin-2 promoter [4], a property that Glis shares with Glis3 [5]
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