E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors

Abstract

The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis.T he vital role of the mammalian lung as a gas-exchange organ is built on the basis of a remarkably elaborate branching sequence during lung development. In the mouse, lung development initiates at embryonic day (E) 9.5 with the formation of two primary lung buds (1, 2). Subsequently, these lung buds elongate and undergo multiple rounds of branching, following a largely stereotyped routine (3, 4). Extensive feedback regulations at the level of transcription have been shown to play central roles in branching (1, 2). In comparison, little is known on how control at the protein level is required for the process in vivo. One study showed that overexpression of SMAD specific ubiquitin protein ligase 1 (Smurf1) led to down-regulation of SMAD family members and a branching defect in lung explant culture (5).The ubiquitin proteasome system is a fundamental molecular mechanism that controls protein homeostasis. Selective degradation of protein substrates by this system involves sequential function of three classes of enzymes: ubiquitin-activating enzyme E1, ubiquitinconjugating enzyme E2, and ubiquitin-protein ligase E3 (6, 7). In a mammalian organism such as the mouse, although there are only a handful of E1 and E2 enzymes that act in the core pathway to mark all protein substrates, there are several hundred E3 ubiquitin ligases, each with a small set of substrates, thus providing specificity (8).E3 ubiquitin ligase Ring finger and WD domain 2 (RFWD2, also known as COP1) was first identified as a regulator in the light sign...

In vitroinduction andin vivoengraftment of lung bud tip progenitor cells derived from human pluripotent stem cells

“…Studies utilizing genetic mouse models have shown that lung branching morphogenesis and proximal-distal patterning are regulated by a series of complex mesenchymal-epithelial interactions that involve multiple signaling events, transcription factors, and dynamic regulation of the physical environment (Domyan and Sun, 2010;Hines and Sun, 2014;Kim and Nelson, 2012;Morrisey and Hogan, 2010;Morrisey et al, 2013;Rawlins, 2010;Rock and Hogan, 2011;Varner and Nelson, 2014). These studies have identified major roles for several signaling pathways in these processes, including Wnt, Fibroblast Growth Factor (Fgf), Bone Morphogenic Protein (Bmp), Sonic Hedgehog (Shh), Retinoic Acid (RA) and Hippo signaling among others (Abler et al, 2009;Bellusci et al, 1997a;Bellusci et al, 1997b;Bellusci et al, 1996;Cornett et al, 2013;Desai et al, 2006;Desai et al, 2004;Domyan et al, 2011;Goss et al, 2009;Harris-Johnson et al, 2009;Herriges et al, 2015;Lange et al, 2015;Lu et al, 2009;Mahoney et al, 2014;Motoyama et al, 1998;Sekine et al, 1999;Shu et al, 2005;Weaver et al, 2000;White et al, 2006;Yin et al, 2011;Yin et al, 2008;Zhang et al, 2016;Zhao et al, 2014). However, due to the complex and intertwined nature of these signaling networks, perturbations in one pathway often affect signaling activity of others (Hines and Sun, 2014;Morrisey et al, 2013;Ornitz and Yin, 2012).…”

“…(RA) and Hippo signaling, among others (Abler et al, 2009;Bellusci et al, 1997a;1997b;1996;Cornett et al, 2013;Desai et al, 2006;2004;Domyan et al, 2011;Goss et al, 2009;Harris-Johnson et al, 2009;Herriges et al, 2015;Lange et al, 2015;Lu et al, 2009;Mahoney et al, 2014;Motoyama et al, 1998;Sekine et al, 1999;Shu et al, 2005;Weaver et al, 2000;White et al, 2006;Yin et al, 2011;Zhang et al, 2016;Zhao et al, 2014). However, due to the complex and intertwined nature of these signaling networks, perturbations in one pathway often affect signaling activity of others (Hines and Sun, 2014;Morrisey et al, 2013;Ornitz and Yin, 2012).…”

Transcription factor Etv5 is essential for the maintenance of alveolar type II cells

“…Conserved in plants, COP1 represses photomorphogenesis until exposure to light causes COP1 to be excluded from the nucleus and apart from its nuclear substrates (19). In mammals, COP1 controls lung-branching morphogenesis (20) and functions as a tumor suppressor by targeting c-JUN or ETV1 for degradation (14,21). How vertebrate CRL4 COP1/DET1 is regulated is less clear.…”

Degradation for better survival? Role of ubiquitination in epithelial morphogenesis

“…Overexpression of Smurf1 promoted ubiquitination and degradation of Smad1 and Smad5 and inhibited lung branching in a BMP4‐dependent manner (Shi et al, ). Further studies on mouse lung morphogenesis identified the E3 ligase RING finger and WD repeat domain 2 (RFWD2) to be a regulator of normal lung branching (Zhang et al, ). Loss of RFWD2 inhibited lung morphogenesis, accompanied by up‐regulation of E26 transformation‐specific (ETS) transcription factors including ets variant 4 (ETV4) and ets variant 5 (ETV5) (Zhang et al, ).…”