A novel homozygous C-terminal deletion in BTG4 causes zygotic cleavage failure and female infertility.

PURPOSE: We aimed to identify pathogenic variants in a female patient with primary infertility and recurrent failure of in vitro fertilization with zygotic cleavage failure. METHODS: The genomic DNA from the affected individual was subjected to whole-exome sequencing and the variant was confirmed by Sanger sequencing. The functional effect of the identified variant was further investigated in 293 T cells. RESULTS: We identified a novel homozygous deletion in BTG4 (c.580_616del) in the affected individual. The deletion results in frameshift and replacement of the last 29 residues (aa195-223) with 66 random amino acids. The mutated amino acid residues are highly conserved among mammalian species. Co-immunoprecipitation in 293 T cells showed that the mutation abolished the interaction between BTG4 and PABPN1L. CONCLUSION: This study conforms previous studies and expands the mutational spectrum of BTG4. Our findings prove the functional importance of the C-terminal of BTG4. BTG4 is a potential diagnostic and therapeutic target for patients suffering from zygotic cleavage failure.

CUL4B renders breast cancer cells tamoxifen-resistant via miR-32-5p/ER-α36 axis.

Tamoxifen (TAM) resistance is a significant clinical challenge in endocrine therapies for estrogen receptor (ER)-positive breast cancer patients. Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through diverse epigenetic mechanisms. However, the role and the underlying mechanisms of CUL4B in regulating drug resistance remain unknown. Here, we showed that CUL4B promotes TAM resistance in breast cancer cells through a miR-32-5p/ER-α36 axis. We found that upregulation of CUL4B correlated with decreased TAM sensitivity of breast cancer cells, and knockdown of CUL4B or expression of a dominant-negative CUL4B mutant restored the response to TAM in TAM-resistant MCF7-TAMR and T47D-TAMR cells. Mechanistically, we demonstrated that CUL4B renders breast cancer cells TAM-resistant by upregulating ER-α36 expression, which was mediated by downregulation of miR-32-5p. We further showed that CRL4B epigenetically represses the transcription of miR-32-5p by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes to promote trimethylation at H3K27 at the promoter of miR-32-5p. Pharmacologic or genetic inhibition of CRL4B/PRC2/HDAC complexes significantly increased TAM sensitivity in breast cancer cells in vitro and in vivo. Taken together, our findings thus establish a critical role for the CUL4B-miR-32-5p-ER-α36 axis in the regulation of TAM resistance and have important therapeutic implications for combined application of TAM and the inhibitors of CRL4B/PRC2/HDAC complex in breast cancer treatment. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aryl hydrocarbon receptor (AhR) regulates adipocyte differentiation by assembling CRL4B ubiquitin ligase to target PPARγ for proteasomal degradation.

Peroxisome proliferator-activated receptor γ (PPARγ) is the central regulator of adipogenesis, and its dysregulation is linked to obesity and metabolic diseases. Identification of the factors that regulate PPARγ expression and activity is therefore crucial for combating obesity. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a known role in xenobiotic detoxification. Recent studies have suggested that AhR also plays essential roles in energy metabolism. However, the detailed mechanisms remain unclear. We previously reported that experiments with adipocyte-specific Cullin 4b (Cul4b)-knockout mice showed that CUL4B suppresses adipogenesis by targeting PPARγ. Here, using immunoprecipitation, ubiquitination, real-time PCR, and GST-pulldown assays, we report that AhR functions as the substrate receptor in CUL4B-RING E3 ubiquitin ligase (CRL4B) complex and is required for recruiting PPARγ. AhR overexpression reduced PPARγ stability and suppressed adipocyte differentiation, and AhR knockdown stimulated adipocyte differentiation in 3T3-L1 cells. Furthermore, we found that two lysine sites on residues 268 and 293 in PPARγ are targeted for CRL4B-mediated ubiquitination, indicating cross-talk between acetylation and ubiquitination. Our findings establish a critical role of AhR in regulating PPARγ stability and suggest that the AhR-PPARγ interaction may represent a potential therapeutic target for managing metabolic diseases arising from PPARγ dysfunction.

ORMDL3 Facilitates the Survival of Splenic B Cells via an ATF6α-Endoplasmic Reticulum Stress-Beclin1 Autophagy Regulatory Pathway.

The genetic association of orosomucoid-like 3 (ORMDL3) with an array of immunoinflammatory disorders has been recently unraveled in multiple ethnic groups, and functional exploration has received attention of the particular relevance of this gene in endoplasmic reticulum stress, lipid metabolism, and inflammatory response. In this study, we demonstrated the upregulation of ORMDL3 in both patients with systemic lupus erythematosus and lupus mice compared with controls. By establishing ORMDL3 knockout mice (Ormdl3-/-), we showed that silencing Ormdl3 in vivo significantly decreased the proportions of mature B lymphocytes and transitional 2B cells in spleen and B1a cells from abdominal cavity perfusion fluid, the secretion of IgG and IgM, and the expression of Baff. Additionally, knockdown of Ormdl3 augmented the apoptosis of total splenic cells and splenic CD19+ B cells but did not affect B cell proliferation and cell cycle. Subsequently, we in vitro and in vivo demonstrated that ORMDL3 potentially mediates the autophagy via the ATF 6-Beclin1 autophagy pathway, and it facilitates the survival of splenic B cells via promoting autophagy and suppressing apoptosis. Taken together, we uncovered a role of ORMDL3 in fine-tuning B cell development and survival, besides highlighting a potential mechanism by which ORMDL3 regulates autophagy via ATF6 pathway.

Ankylosing spondylitis: analysis of gene-gene interactions between IL-12ß, JAK2, and STAT3 in Han Chinese and Algerian cohorts.

INTRODUCTION: Association studies have recently identified the importance of new genetic variants for ankylosing spondylitis (AS) in several populations. Our aim was to confirm associations of variants within genes involved in the IL-23 signalling pathway with AS in two ethnically different populations: Han Chinese and Algerian. MATERIAL AND METHODS: Two case-control studies were performed in separate cohorts: Han Chinese (430 AS patients and 580 controls) and Algerian (130 AS patients and 120 controls). We genotyped four single nucleotide polymorphisms (SNPs): rs3212227 (or +1188A/C) and rs6887695 in IL-12ß, rs7857730 in JAK2, and rs2293152 in STAT3, using TaqMan SNP genotyping assays. Gene-gene interaction analyses were also tested by logistic regression and multifactor dimensionality reduction (MDR). RESULTS: Statistical analysis revealed a difference in allele frequencies between AS patients and controls for rs321222 in the IL-12ß gene in both the Han Chinese (p = 0.005) and the Algerian (p = 0.031) cohorts. Two other associations were reported with JAK2 rs7857730 in the Han Chinese (allelic p = 0.014) cohort and STAT3 rs2293152 in the Algerian (allelic p = 0.006) cohort. Moreover, logistic regression analyses showed a number of significant combinations within the two populations, and the gene-gene epistasis effects in AS were also confirmed by MDR. CONCLUSIONS: Our findings have confirmed the association between genes in IL-23 signalling pathway and the pathogenesis of AS. This association was particularly novel in both Han Chinese and Algerian populations with the 3' untranslated region (3'UTR) variant rs3212227 (or +1188A/C) of IL-12ß. The gene-gene interaction models in this pathway may thus increase the risk of AS in these populations.

HBV suppresses ZHX2 expression to promote proliferation of HCC through miR-155 activation.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.

Whole-Genome Linkage Analysis with Whole-Exome Sequencing Identifies a Novel Frameshift Variant in NEFH in a Chinese Family with Charcot-Marie-Tooth 2: A Novel Variant in NEFH for Charcot-Marie-Tooth 2.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is the most common neurodegenerative disorder of the peripheral nervous system. More than 50 genes/loci were found associated with the disease. We found a family with autosomal-dominant CMT2. OBJECTIVE: To reveal the pathogenic gene of the family and further investigate the function of the variant. METHODS: DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members. Neurofilament light polypeptide and wild-type or mutant neurofilament heavy polypeptide (NEFH) were co-transfected into SW13 (vim-) cells. The nefh-knockdown zebrafish model was produced by using morpholino antisense oligonucleotides. RESULTS: We identified a novel insertion variant (c.3057insG) in NEFH in the family. The variant led to the loss of a stop codon and an extended 41 amino acids in the protein. Immunofluorescence results revealed that mutant NEFH disrupted the neurofilament network and induced aggregation of NEFH protein. Knockdown of nefh in zebrafish caused a slightly or severely curled tail. The motor ability of nefh-knockdown embryos was impaired or even absent, and the embryos showed developmental defects of axons in motor neurons. The abnormal phenotype and axonal developmental defects could be rescued by injection of human wild-type but not human mutant NEFH mRNA. CONCLUSIONS: We identified a novel stop loss variant in NEFH that is likely pathogenic for CMT2, and the results provide further evidence for the role of an aberrant assembly of neurofilament in CMT.

Zebrafish cul4a, but not cul4b, modulates cardiac and forelimb development by upregulating tbx5a expression.

CUL4A and CUL4B are closely related cullin family members and can each assemble a Cullin-RING E3 ligase complex (CRL) and participate in a variety of biological processes. While the CRLs formed by the two cullin members may have common targets, the two appeared to have very different consequences when mutated or disrupted in mammals. We here investigated the roles of cul4a and cul4b during zebrafish embryogenesis by using the morpholino knockdown approach. We found that cul4a is essential for cardiac development as well as for pectoral fin development. Whereas cul4a morphants appeared to be unperturbed in chamber specification, they failed to undergo heart looping. The failures in heart looping and pectoral fin formation in cul4a morphants were accompanied by greatly reduced proliferation of cardiac cells and pectoral fin-forming cells. We demonstrated that tbx5a, a transcription factor essential for heart and limb development, is transcriptionally upregulated by cul4a and mediates the function of cul4a in cardiac and pectoral fin development. In contrast to the critical importance of cul4a, cul4b appeared to be dispensable for zebrafish development and was incapable of compensating for the loss of cul4a. This work provides the first demonstration of an essential role of cul4a, but not cul4b, in cardiac development and in the regulation of tbx5a in zebrafish. These findings justify exploring the functional role of CUL4A in human cardiac development.

Lack of CUL4B leads to increased abundance of GFAP-positive cells that is mediated by PTGDS in mouse brain.

Astrocytes are the most abundant cell type in the mammalian brain and are important for the functions of the central nervous system. Glial fibrillary acidic protein (GFAP) is regarded as a hallmark of mature astrocytes, though some GFPA-positive cells may act as neural stem cells. Missense heterozygous mutations in GFAP cause Alexander disease that manifests leukodystrophy and intellectual disability. Here, we show that CUL4B, a scaffold protein that assembles E3 ubiquitin ligase, represses the expression of GFAP in neural progenitor cells (NPCs) during brain development. Lack of Cul4b in NPCs in cultures led to increased generation of astrocytes, marked by GFAP and S100ß. The GFAP+ cells were also found to be more abundant in the brains of nervous system-specific Cul4b knockout mice in vivo. Moreover, we demonstrated that the increased generation of GFAP+ cells from Cul4b-null NPCs was mediated by an upregulation of prostaglandin D2 synthase PTGDS. We showed that the increased GFAP expression can be attenuated by pharmacological inhibition of the PTGDS enzymatic activity or by shRNA-mediated knockdown of Ptgds. Importantly, exogenously added PTGDS could promote the generation of GFAP+ cells from wild-type NPCs. We further observed that Ptgds is targeted and repressed by the CUL4B/PRC2 complex. Together, our results demonstrate CUL4B as a negative regulator of GFAP expression during neural development.

miR-155 Deficiency Ameliorates Autoimmune Inflammation of Systemic Lupus Erythematosus by Targeting S1pr1 in Faslpr/lpr Mice.

MicroRNA-155 (miR-155) was previously found involved in the development of systemic lupus erythematosus (SLE) and other autoimmune diseases and the inflammatory response; however, the detailed mechanism of miR-155 in SLE is not fully understood. To explore the in vivo role of miR-155 in the pathogenesis of SLE, miR-155-deficient Fas(lpr/lpr) (miR-155(-/-)Fas(lpr/lpr)) mice were obtained by crossing miR-155(-/-) and Fas(lpr/lpr) mice. Clinical SLE features such as glomerulonephritis, autoantibody levels, and immune system cell populations were compared between miR-155(-/-)Fas(lpr/lpr) and Fas(lpr/lpr) mice. Microarray analysis, RT-PCR, Western blot, and luciferase reporter gene assay were used to identify the target gene of miR-155. miR-155(-/-)Fas(lpr/lpr) mice showed milder SLE clinical features than did Fas(lpr/lpr)mice. As compared with Fas(lpr/lpr) mice, miR-155(-/-)Fas(lpr/lpr) mice showed less deposition of total IgA, IgM, and IgG and less infiltration of inflammatory cells in the kidney. Moreover, the serum levels of IL-4 and IL-17a, secreted by Th2 and Th17 cells, were lower in miR-155(-/-)Fas(lpr/lpr) than Fas(lpr/lpr) mice; the CD4(+)/CD8(+) T cell ratio was restored in miR-155(-/-)Fas(lpr/lpr) mice as well. Sphingosine-1-phosphate receptor 1 (S1PR1) was found as a new target gene of miR-155 by in vitro and in vivo studies; its expression was decreased in SLE patients and Fas(lpr/lpr) mice. miR-155(-/-)Fas(lpr/lpr) mice are resistant to the development of SLE by the regulation of the target gene S1pr1. miR-155 might be a new target for therapeutic intervention in SLE.

Aberrant RNA splicing as the molecular basis of some pathogenic variants.

Identification and correct classification of disease-associated mutations are essential for molecular diagnosis and clinical management of many genetic disorders. Although next-generation sequencing has greatly accelerated the detection of nucleotide changes, the biological interpretation of most variants has become a real challenge. Moreover, attention is typically paid to protein-coding changes and the potential impact of exonic variants on RNA splicing is often ignored. There is increasing evidence showing that disease-causing aberrant RNA splicing is more widespread than currently appreciated. Here, we review the major types of the variants involved in RNA splicing and the approaches used to identify and characterize these variants. We hope to provide a reference for evaluation of the effects of mutations on diseases.

CRL4B interacts with and coordinates the SIN3A-HDAC complex to repress CDKN1A and drive cell cycle progression.

CUL4B, a scaffold protein that assembles the CRL4B ubiquitin ligase complex, participates in the regulation of a broad spectrum of biological processes. Here, we demonstrate a crucial role of CUL4B in driving cell cycle progression. We show that loss of CUL4B results in a significant reduction in cell proliferation and causes G1 cell cycle arrest, accompanied by the upregulation of the cyclin-dependent kinase (CDK) inhibitors (CKIs) p21 and p57 (encoded by CDKN1A and CDKN1C, respectively). Strikingly, CUL4B was found to negatively regulate the function of p21 through transcriptional repression, but not through proteolysis. Furthermore, we demonstrate that CRL4B and SIN3A-HDAC complexes interact with each other and co-occupy the CDKN1A and CDKN1C promoters. Lack of CUL4B led to a decreased retention of SIN3A-HDAC components and increased levels of acetylated H3 and H4. Interestingly, the ubiquitylation function of CRL4B is not required for the stable retention of SIN3A-HDAC on the promoters of target genes. Thus, in addition to directly contributing to epigenetic silencing by catalyzing H2AK119 monoubiquitylation, CRL4B also facilitates the deacetylation function of SIN3A-HDAC. Our findings reveal a coordinated action between CRL4B and SIN3A-HDAC complexes in transcriptional repression.

CUL4B activates Wnt/ß-catenin signalling in hepatocellular carcinoma by repressing Wnt antagonists.

Activation of Wnt/ß-catenin signalling is frequently observed in many types of cancer including hepatocellular carcinoma (HCC). We recently reported that cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumours and contributes to epigenetic silencing of tumour suppressors. In this study, we characterized the function of CUL4B in HCC and investigated whether CUL4B is involved in the regulation of Wnt/ß-catenin signalling. CUL4B and ß-catenin were frequently up-regulated and positively correlated in HCC tissues. CUL4B activated Wnt/ß-catenin signalling by protecting ß-catenin from GSK3-mediated degradation, achieved through CUL4B-mediated epigenetic silencing of Wnt pathway antagonists. Knockdown of CUL4B resulted in the up-regulation of Wnt signal antagonists such as DKK1 and PPP2R2B. Simultaneous knockdown of PPP2R2B partially reversed the down-regulation of ß-catenin signalling caused by CUL4B depletion. Furthermore, CRL4B promoted the recruitment and/or retention of PRC2 at the promoters of Wnt antagonists and CUL4B knockdown decreased the retention of PRC2 components as well as H3K27me3. Knockdown of CUL4B reduced the proliferation, colony formation, and invasiveness of HCC cells in vitro and inhibited tumour growth in vivo, and these effects were attenuated by introduction of exogenous ß-catenin or simultaneous knockdown of PPP2R2B. Conversely, ectopic expression of CUL4B enhanced the proliferation and invasiveness of HCC cells. We conclude that CUL4B can up-regulate Wnt/ß-catenin signalling in human HCC through transcriptionally repressing Wnt antagonists and thus contributes to the malignancy of HCC.

DNM2 mutations in Chinese Han patients with centronuclear myopathy.

Centronuclear myopathy (CNM) is a congenital myopathy characterized by an abnormally high number of muscle fibers with centrally located nuclei. Autosomal-dominant centronuclear myopathy-1 (CNM1) results from mutations in the dynamin 2 gene (DNM2) and accounts for approximately 50 % of all CNM cases. Up to now, around 35 mutations of DNM2 gene have been identified in CNM; however, the underlying molecular mechanism of DNM2 mutation in the pathology of CNM remains elusive, and the standard clinical characteristics and the genotype-phenotype correlation of DNM2 gene mutation with CNM have not yet been defined. Here, we report the clinical characteristics, molecular diagnosis strategy, and DNM2 gene mutations of four Chinese Han patients with CNM. Congenital myopathy-targeted next-generation sequencing (NGS) was applied to sequence the regions of the genome that contain all the coding regions of all known CNM genes and other congenital myopathy genes. We found potential DNM2 mutations in all four of the patients. Further targeted Sanger DNA sequencing of DNM2 found the 1106G>A (p.R369Q) mutation in patients 1 and 2, the c.1393C>T (p.R465W) mutation in patient 3, and the c.1565G>A (p.R522H) mutation in patient 4, all of which were reported previously to be causative mutations of DNM2-related CNM. Our results suggest that the combination of targeted NGS and Sanger sequencing is an effective, rapid, and reliable strategy for the molecular diagnosis of CNM and other genetically heterogeneous disorders.

Identification and functional analysis of a SLC33A1: c.339T>G (p.Ser113Arg) variant in the original SPG42 family.

Using whole-exome sequencing, we surveyed all the potential pathogenic variants in an SPG42 family and found five SNPs and four indels that are shared by two patients and lie in the mapped region. Two variants, SLC33A1 p.Ser113Arg and VEPH1 p.Gln433His, cosegregated with the disease. However, VEPH1 p.Gln433His was predicted to be tolerated, thus leaving SLC33A1 p.Ser113Arg as the most plausible causal variant in this family. We found that the phosphorylated SMAD1/5/8 (P-SMAD1/5/8) and BMP receptor type 1A (BMPR1A) were substantially upregulated in fibroblasts derived from an SPG42 individual. Slc33a1 knockdown zebrafish, which exhibited defects in morphology and axon outgrowth, also showed a significant elevation in the level of P-smad1/5/8. While the phenotypes in slc33a1 knockdown zebrafish could be rescued by human wild-type SLC33A1 mRNA, this rescuing effect was diminished by coinjected mutant mRNA encoding p.Ser113Arg, indicating that p.Ser113Arg variant acts in a dominant-negative manner. Importantly, pharmacological blockade of BMPR1 activity by dorsomorphin could efficiently rescue the phenotypic defects in slc33a1 knockdown zebrafish. These results indicate that SLC33A1 can negatively regulate BMP signaling.

Whole-exome sequencing identifies a variant in TMEM132E causing autosomal-recessive nonsyndromic hearing loss DFNB99.

Autosomal-recessive nonsyndromic hearing loss (ARNSHL) features a high degree of genetic heterogeneity. Many genes responsible for ARNSHL have been identified or mapped. We previously mapped an ARNSHL locus at 17q12, herein designated DFNB99, in a consanguineous Chinese family. In this study, whole-exome sequencing revealed a homozygous missense mutation (c.1259G>A, p.Arg420Gln) in the gene-encoding transmembrane protein 132E (TMEM132E) as the causative variant. Immunofluorescence staining of the Organ of Corti showed Tmem132e highly expressed in murine inner hair cells. Furthermore, knockdown of the tmem132e ortholog in zebrafish affected the mechanotransduction of hair cells. Finally, wild-type human TMEM132E mRNA, but not the mRNA carrying the c.1259G>A mutation rescued the Tmem132e knockdown phenotype. We conclude that the variant in TMEM132E is the most likely cause of DFNB99.

Idebenone protects against oxidized low density lipoprotein induced mitochondrial dysfunction in vascular endothelial cells via GSK3ß/ß-catenin signalling pathways.

The early stages of the atherosclerotic process are initiated by accumulation of oxidized low-density lipoprotein (oxLDL) and damage to the structure or function of the endothelium. Antioxidant supplementation may be a plausible strategy to prevent atherosclerotic disease by quenching excessive reactive oxidative species. In the present study, we demonstrated that idebenone at suitable concentrations significantly prevented oxLDL-induced endothelial dysfunction. The underlying mechanisms of idebenone included inhibition of oxidative damage, suppression of the down-regulation of Bcl-2 and up-regulation of Bax and cleaved caspase-3 in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL. Moreover, idebenone pretreatment inhibited oxLDL-mediated HUVECs damage by attenuating lipid peroxidation and promoting SOD activity. Finally, pro-incubation with idebenone inhibited mitochondrial dysfunction induced by oxLDL through the mitochondrial-dependent apoptotic pathway and GSK3ß/ß-catenin signalling pathways. In summary, idebenone is a promising agent in the treatment or prevention of atherosclerosis via inhibiting oxidative stress and improving mitochondrial function.

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.

CUL4B mutations impair human cortical neurogenesis through PP2A-dependent inhibition of AKT and ERK.

Mutation in CUL4B gene is one of the most common causes for X-linked intellectual disability (XLID). CUL4B is the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complex. While the roles of CUL4B in cancer progression and some developmental processes like adipogenesis, osteogenesis, and spermatogenesis have been studied, the mechanisms underlying the neurological disorders in patients with CUL4B mutations are poorly understood. Here, using 2D neuronal culture and cerebral organoids generated from the patient-derived induced pluripotent stem cells and their isogenic controls, we demonstrate that CUL4B is required to prevent premature cell cycle exit and precocious neuronal differentiation of neural progenitor cells. Moreover, loss-of-function mutations of CUL4B lead to increased synapse formation and enhanced neuronal excitability. Mechanistically, CRL4B complex represses transcription of PPP2R2B and PPP2R2C genes, which encode two isoforms of the regulatory subunit of protein phosphatase 2 A (PP2A) complex, through catalyzing monoubiquitination of H2AK119 in their promoter regions. CUL4B mutations result in upregulated PP2A activity, which causes inhibition of AKT and ERK, leading to premature cell cycle exit. Activation of AKT and ERK or inhibition of PP2A activity in CUL4B mutant organoids rescues the neurogenesis defect. Our work unveils an essential role of CUL4B in human cortical development.