Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

CUL4B ubiquitin ligase in mouse development: A model for human X-linked mental retardation syndrome?

CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.CUL4 is a member of the cullin-RING ubiquitin ligase family, the largest E3 ligase family, which appears to account for ∼20% of total protein degradation by the ubiquitin-proteasome system^1,2,3. CUL4 is conserved during evolution from yeast to human. In yeast, CUL4 encodes a single gene, but mammalian cells express two closely related paralogs, CUL4A and CUL4B with about 82% sequence identity. CUL4A and CUL4B assemble structurally similar E3 complexes through binding to an adaptor protein (DDB1) and a substrate receptor protein (DCAF) at the N-terminus, and a RING protein RBX1 at the C-terminus (Figure 1), and share functional redundancy in targeting substrates such as p21 and Cdt1 for ubiquitination and degradation^1,2. The Cul4a-null mice are viable and display no abnormal development and growth phenotypes, likely due to functional compensation from Cul4b^4,5. The only phenotype associated with Cul4a abrogation is the reproductive defects seen with male but not female mice, resulting from differential non-overlapping expression patterns of the two Cul4 genes during male meiosis⁶. On the other hand, germline deletion of Cul4b resulted in embryonic lethality around E9.5⁷, indicating a unique function of Cul4b that cannot be compensated by Cul4a during embryogenesis.Open in a separate windowFigure 1Differential expression of Cul4a and Cul4b in the embryo proper and extra embryonic tissues determines their fate. Before implantation, both Cul4a and Cul4b are expressed in the blastocyst. Following implantation, Cul4a is expressed in the embryo proper, but not in extra-embryonic tissues. Upon Cul4b deletion, p21 accumulates in extra-embryonic tissues to induce G2/M arrest and eventually embryonic death due to degeneration of extra-embryonic tissues. Expression of Cul4a in embryo prevents p21 accumulation and subsequent embryonic death.Mental retardation (MR) affects approximately 1%-3% of the population and is about 30% more common in males than in females⁸, suggesting a causal relationship with gene mutations on the X chromosome. To date, mutations in about 100 genes have been identified in X-linked MR (XLMR), much more than those found on autosomes⁹. In 2007, two independent groups reported that mutations of CUL4B (Xq24) ubiquitin ligase gene are associated with XLMR^10,11. CUL4B-deficient patients display a syndrome of delayed puberty, moderate short stature, hypogonadism, relative macrocephaly, central obesity, fine intention tremor, brachydactyly, and large tongue^10,11. Similarly, the neuronal and developmental deficiencies found in XLMR patients with CUL4B mutations are not compensated by CUL4A. The studies of the molecular pathogenesis of human XLMR are lagging partly due to the lack of an animal model for the disease.In the most recent study published in Cell Research, Zhou and coworkers¹² attempted to generate conditional Cul4b knockout mice with targeted deletion of Cul4b at exons 4 and 5, giving rise to a non-functional Cul4b fragment lacking both the DDB1-binding domain and the cullin homology domain for RBX1 recruitment. The chicken-actin (CAG)-Cre was used, which drives Cre-mediated recombination at the early zygote stage, leading to Cul4b deletion in both the embryo proper and extra-embryonic tissues. Like human CUL4B, the mouse Cul4b is also located on the X-chromosome. Intercrossing of male CAG-Cre with female Cul4b^fl/+ revealed that hemizygous deletion of Cul4b causes embryonic lethality. No embryos with the genotype of Cul4b^−/y survived beyond E9.5. Interestingly, the heterozygous Cul4b^+/− embryos also die in the uterus before E13.5, suggesting that the paternal X chromosome undergoes imprinted inactivation with only trace amount, if any, of Cul4b expression remaining in extra-embryonic tissues. Detailed analysis of dissected embryos revealed that dying Cul4b^+/− embryos (E12.5) lack blood supply from the yolk sacs, whereas the Cul4b^−/y embryos (E8.5) showed remarkable reduction in proliferation with growth arrest at G2/M and enhanced apoptosis. The authors went on and investigated why Cul4a failed to compensate the loss of Cul4b, and found a dynamic expression pattern, differing between two forms, during early embryonic development. Prior to implantation, both Cul4 proteins are detectable in the blastocysts. Shortly after implantation, while both forms are expressed in the embryo proper, only Cul4b is expressed in the extra-embryonic tissues. Thus, upon Cul4b deletion, extra-embryonic tissues without Cul4a compensation degenerate, eventually leading to embryonic death. Consistently, when the authors deleted Cul4b in the epiblast using the Sox2-Cre (targeted Cul4b deletion in embryos proper only), viable Cul4b-null mice are produced likely due to Cul4a compensation. Thus, Cul4b is essential for the development of extra-embryonic tissues, but is dispensable for embryogenesis itself.To study the potential underlying mechanism(s) of embryonic lethality upon Cul4b deletion in extra-embryonic tissues, the authors used an extra-embryonic cell line (XEN). Cul4b knockdown induced a remarkable cell cycle arrest at the G2/M phase, consistent with observation made in Cul4b-null embryos, and robust accumulation of p21, a universal inhibitor of cyclin dependent kinase and a known substrate of Cul4¹. To determine whether accumulated p21 is responsible for the G2/M arrest, the authors simultaneously knocked down both Cul4b and p21 in XEN cells and observed a partial abrogation of growth arrest, suggesting that p21 plays a causal role, at least in part. Unfortunately, due to unavailability of anti-mouse p21 antibody specific for immunohistochemical staining, the authors were not able to show if p21 is indeed accumulated in extra-embryonic tissues upon Cul4b deletion. However, whether p21 indeed plays a causal role in embryonic death upon Cul4b deletion can be unequivocally determined by a rescuing experiment in which simultaneous deletion of p21 should abrogate or at least delay embryonic lethality, if it is causal. Nevertheless, the study by Zhou''s group can be summarized as follows. Before implantation, both Cul4a and Cul4b ubiquitin ligases are expressed in the blastocyst (inner cell mass and trophoblast cells). Following embryo implantation, while Cul4b is expressed in both the embryo proper and extra embryonic tissues, Cul4a is only expressed in the embryo proper. The CAG-Cre-driven Cul4b deletion (in both the embryo proper and extra-embryonic tissues) causes significant p21 accumulation in Cul4a non-expressing extra-embryonic tissues, resulting in G2/M arrest, followed by embryonic death due to degeneration of extra-embryonic tissues. On the embryo side, Cul4b deletion has no detrimental consequence, benefiting from the compensatory effect of Cul4a for p21 targeting. The same holds true when Cul4b is deleted driven by embryonic specific Sox2-Cre (Figure 1).It is noteworthy that the studies by Zhou''s group revealed two distinct differences between Cul4b KO mice and CUL4B-associated XLMR patients. First, Cul4b deletion at the zygote stage causes embryonic lethality, whereas XLMR patients with CUL4B mutations live to adulthood. Second, the Cul4b-null allele cannot be transmitted from the mother to the offspring, whereas human XLMR patients inherit X-linked CUL4B mutations from their mothers. Nevertheless, viable Cul4b-null mice (upon epiblast ablation by Sox2-Cre) provide the first mouse model for mechanistic study of human XLMR diseases associated with CUL4B mutations in the following three aspects:First, as noted earlier, human CUL4B XLMR patients have multiple neuronal and developmental defects. An obvious follow-up study will be to use this mouse model for neurological and behavioral analyses to determine whether Cul4b-null mice indeed present some of human XLMR symptoms.Second, this model can also be used to validate whether accumulation of Cul4b substrates during various stages of brain development indeed plays a pathogenic role and contributes to the clinical symptoms of XLMR patients. For instance, WDR5, a recently identified gene affecting general cognitive ability¹³, was found to be a novel nuclear substrate of CUL4B, but not CUL4A¹⁴. Investigation into whether WDR5 is abnormally accumulated upon Cul4b deletion in vivo would rule in or rule out its potential association with human XLMR, although it was not the case in this study using an extra-embryonic cell line in vitro.Third, the viability of Cul4b-null mice upon epiblast-specific deletion provides opportunities to study neuronal specific ablation of Cul4b in association with the pathogenesis of CUL4B-associated XLMR. For example, Cul4b is expressed at high levels in the hippocampus and cerebrum of mouse brains; both regions are affected in MR patients¹⁵. Thus, the use of Cre mouse lines that target the deletion of Cul4b in the entire brain, selected brain areas, or specific neuronal cells in both spatial and temporal manners¹⁶ would reveal potential contributions of particular regions and cell types to the development and symptoms of CUL4B-associated XLMR.A number of questions that warrant future investigation remain unanswered. First, in addition to p21, what are the other Cul4B substrates, which also contribute to degeneration of extra-embryonic tissues upon Cul4b deletion, since simultaneous deletion of p21 only partially rescues the growth defects? Second, besides the difference in tissue/cell specific expression seen in this study, are Cul4a and Cul4b targeting a unique set of substrates non-redundantly, thus differentiating their physiological functions? A related question will be why CUL4A cannot compensate for the loss of CUL4B in CUL4B-associated XLMR patients? Third, what is the pathogenic mechanism for CUL4B-associated XLMR? Is it mainly due to pathological accumulation of many CUL4B substrates? Answers to these questions may offer insights into potential therapeutic strategies for the treatment of CUL4B-associated XLMR patients.In summary, the findings reported by Zhou''s group provide the first convincing evidence that demonstrates an essential role of Cul4b in the development of extra-embryonic tissues during mouse embryogenesis. The viable Cul4b conditional knockout mice, generated in this study, may serve as the first mouse model for future mechanistic studies of neuronal and behavioral deficiencies of human XLMR associated with CUL4B mutations. We look forward to more exciting discoveries of how Cul4b deficiency leads to the development of XLMR in years to come.     

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8^AN015 or CSN5^null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

Loss of the Mono-ADP-ribosyltransferase,Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp^−/− mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp^+/+ mice survived the 30-day treatment. Dioxin-treated Tiparp^−/− mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.     

DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

The human inhibitor of Bruton''s tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3^IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3^IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3^IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3^IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.     

Circ_0021573 acts as a competing endogenous RNA to promote the malignant phenotypes of human ovarian cancer cells

Circular RNAs (circRNAs) have been reported to be implicated in the tumorigenesis and progression of ovarian cancer. Here, the study was designed to explore the activity of human circ_0021573 in ovarian cancer pathogenesis and its regulation through the competing endogenous RNA (ceRNA) crosstalk. Circ_0021573, microRNA (miR)? 936, and cullin 4B (CUL4B) were quantified by qRT-PCR and western blot. Cell proliferation ability was detected by XTT, 5-Ethynyl-2′-Deoxyuridine (EdU), and colony formation assays. Cell apoptosis, migration, and invasion were assessed by flow cytometry, wound-healing, and transwell assays, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-936 and circ_0021573 or CUL4B 3′UTR. Xenograft studies were applied to assess the role of circ_0021573 in tumor growth. Our data showed that circ_0021573 expression is enhanced in human ovarian cancer. Inhibition of circ_0021573 impedes cell proliferation, migration, and invasion and promotes apoptosis in vitro, as well as diminishes tumor growth in vivo. Mechanistically, circ_0021573 contains a miR-936 binding site, and miR-936 is a relevant mediator of circ_0021573 regulation. MiR-936 direct targets and inhibits CUL4B. MiR-936-mediated suppression of CUL4B hinders cell proliferation, migration, and invasion and accelerates apoptosis in vitro.. These data suggested that circ_0021573 might promote the malignant phenotypes of ovarian cancer cells by functioning as a ceRNA for miR-936 to induce CUL4B, which provided a promising target for the prevention and inhibition of ovarian cancer.     

Generation of a 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahrd mouse line harboring the poor-affinity aryl hydrocarbon receptor

Herein, we describe generation of the hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mouse line, which carries human functional CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a1 and Cyp1a2 genes, in a (>99.8%) background of the C57BL/6J genome and harboring the poor-affinity aryl hydrocarbon receptor (AHR) from the DBA/2J mouse. We have characterized this line by comparing it to our previously created hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 line—which carries the same but has the high-affinity AHR of the C57BL/6J mouse. By quantifying CYP1A1 and CYP1A2 mRNA in liver, lung and kidney of dioxin-treated mice, we show that dose-response curves in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mice are shifted to the right of those in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 mice—similar to, but not as robust as, dose-response curves in DBA/2J versus C57BL/6J mice. This new mouse line is perhaps more relevant than the former to human risk assessment vis-à-vis human CYP1A1 and CYP1A2 substrates, because poor-affinity rather than high-affinity AHR occurs in the vast majority of the human population.