Establishment And Preliminary Application Of IPSCs With CUL4B Loss-of-function Mutations

As a member of the Cullin family,the scaffold protein CUL4B participates in the formation of the Cullin 4B-RING E3 ubiquitin ligase complexes（Cullin 4B-RING E3 ligases,CRL4Bs）.CRL4B not only mediates protein degradation through polyubiquitination of substrate proteins,but also catalyzes monoubiquitination of H2AK119 to play an epigenetic regulatory role by coordinating multiple transcriptional inhibitory complexes.In 2007,a British research group and our research group successively found that mutations in CUL4B gene cause X-linked mental retardation syndrome.The subsequent studies have confirmed that CUL4B gene mutation is one of the three most common causes of X-linked mental retardation syndrome.Analysis of the reported clinical phenotypes of patients with CUL4B mutations revealed that mental retardation,short stature,aphasia and epilepsy were the most common clinical manifestations.Using gene knockout mice model,it was found that mice lacking CUL4B in the central nervous system showed impaired learning and memory,less PV-positive GABAergic interneurons in hippocampus,reduced dendritic branches and cell size of hippocampal neurons,and abnormal dendritic spine density and morphology.However,the mechanism of CUL4B mutation causing phenotypes such as mental retardation is still unclear.Induced pluripotent stem cells（iPSCs）are embryonic stem cell-like cells with self-renewal and multidirectional differentiation potential,derived from reprogramming of terminally differentiated cells.iPSCs can be derived from a large variety of donor cells and are easy to amplify and differentiate into various functional cells.Since iPSCs technology became available,patients-derived iPSCs and the directional differentiation cell models have been used in the research of a variety of genetic diseases,especially in neural development and neurodegenerative diseases,because diseased tissue was difficult to acquire,and animal models like mice could not simulate well the pathological characteristics of human diseaseIn order to study the mechanism of X-linked mental retardation syndrome caused by CUL4B loss-of-function mutations,this project intends to construct a CUL4B mutation-specific iPSCs model to analyze the effects of CUL4B deletion on iPSCs production,iPSCs proliferation,and neural progenitor cells induced by iPSCs,and lay the foundation for understanding the pathogenic mechanism of CUL4B mutation.Part ⅠConstruction and preliminary application of the induced pluripotent stem cells from Cul4b-deficient miceAlthough many studies have shown that CUL4B mutation is one of the three main causes of X-linked mental retardation syndrome,and mice lacking Cul4b exhibited learning and memory impairment,the mechanism by which CUL4B mutations cause cognitive impairment in X-linked mental retardation syndrome is still unclear.iPSCs are a good platform for studying the pathogenesis of genetic diseases,especially neurodevelopmental diseases.Therefore,this project intends to establish a CUL4B mutation-specific iPSCs model and explore the mechanism of mental retardation syndrome caused by CUL4B mutationStudies have shown that changes in epigenetic modifications can affect somatic cell reprogramming.The CUL4B complex can catalyze the monoubiquitination of histone H2AK119 to change the binding status of multiple transcription repressor complexes in the promoter regions of specific genes.Therefore,we speculated that CUL4B loss-of-function mutations may affect the efficiency of somatic cell reprogramming.In the first part of this study,we analyzed the effect of deletion of Cul4b on the reprogramming of mouse embryonic fibroblast cells（MEFs）and the function of the miPSCs,and obtained the following results:1.By mating Cul4bfn/fn female mice with Sox2-Cre+/-male mice,we obtained Cul4b-deficient and control MEFs.EdU incorporation experiments showed that MEFs lacking CUL4B have proliferation defects2.We transfered the four transcription factors Oct4,Sox2,c-Myc and Klf4 into the control and Cul4b-deficient MEFs to induce miPSCs of two genotypes3.Characterization of miPSCs:1)Analysis of pluripotent stem cell markers.Immunofluorescence was used to detect the expression of pluripotent markers OCT4,SOX2 and SSEA1.All three markers were expressed in the obtained miPSCs.Real-time qPCR showed that in the established miPSCs,the endogenous pluripotent genes were activated,while exogenous pluripotent genes were silenced;2)Analysis of the differentiation potential of the three germ layers.The important characteristic of pluripotent stem cells is that they can differentiate into a variety of functional cells.In order to analyze whether CUL4B deletion affects the differentiation potential of iPSCs,we analyzed the three germ layers differentiation potential of two genotypes of miPSCs in vitro and in vivo.Wild-type and Cul4b-deficient miPSCs were cultured into embryoid bodies and differentiated,and the expression of specific markers in endoderm,mesoderm and ectoderm could be detected;3)Karyotype analysis showed that the wild-type and Cul4b-deficient miPSCs both had normal karyotypes.4.Lack of CUL4B increased the reprogramming efficiency of MEFs.The same number of wild-type and Cul4b-deficient MEFs were induced to miPSCs separately.Alkaline phosphatase（AP）staining was performed after 9-day induction.The number of AP positive clones formed by Cul4b-deficient cells was significantly higher than that formed by the wild-type cells.RT-qPCR and Western Blot showed that the deletion of Cul4b increased endogenous Nanog gene expression,indicating that CUL4B played an inhibitory role in the reprogramming process of miPSCs.5.CUL4B regulates the reprogramming of MEFs by regulating the expression of E-Cadherin:1)We used Western Blot to analyze the expression of the known signal pathway molecules that regulate somatic cell reprogramming.We found that the absence of CUL4B did not affect the Wnt pathway,BMP pathway and H3K9me3.However,it significantly up-regulated mRNA and protein levels of E-Cadherin;2)We further found that overexpression of CUL4B resulted in down-regulation of E-Cadherin expression and reduced reprogramming efficiency,indicating that CUL4B negatively regulated the expression of E-Cadherin,and inhibited reprogramming;3)To determine whether CUL4B inhibits MEFs reprogramming by regulating the expression of E-Cadherin,we knocked down Cdhl in Cul4b-deficient MEFs to reduce E-Cadherin to a level similar to that of the wild type.We found that it could attenuate the elevated reprogramming efficiency caused by loss of CUL4B,confirming that CUL4B inhibited reprogramming by suppressing the expression of E-Cadherin;4)In order to investigate how CUL4B regulates Cdhl gene expression,we used ChIP experiments to analyze the binding of CUL4B complex in the promoter region of Cdhl gene.We found that CUL4B and another component of the complex DDB1 both bind to the promoter of Cdh1.The catalytic product of CRL4B,H2AK119ub1,was enriched in the promoter of Cdh1.In addition,depletion of CUL4B reduced the enrichment of H2AK119ub1 and the interaction complex PRC2 catalytic product H3K27me3 in the promoter of Cdh1,suggesting that CUL4B complex inhibited the transcription of Cdh1 gene by catalyzing H2AK119ub1.6.Absence of CUL4B inhibited the proliferation of miPSCs.The proliferation of miPSCs was detected by EdU incorporation experiments.The proliferation rate of Cul4b null miPSCs was found to be significantly reduced compared to that of wild-type.When wild-type and Cul4b null miPSCs were mix cultured in equal proportions,the number of Cul4b null miPSCs gradually decreased and disappeared quickly.Cell cycle analysis showed that Cul4b knockout resulted in G1 phase arrest.Protein analysis showed that deletion of CUL4B caused p21 up-regulation.7.miPSCs were induced to differentiate into neural progenitor cells（NPCs）.EdU incorporation experiment was used to evaluate the proliferation rate of wild-type and Cul4b null NPCs.We found that the absence of CUL4B reduced the proliferation.Flow cytometry revealed that CUL4B deficiency led to G1 arrest in NPCs,accompanied by p21 accumulation.8.NPCs were induced to differentiate toward neurons and glial cells.CUL4B deletion led to reduced neuronal differentiation from NPCs,less branches of neurons and increased NOTCH1 expression.Inhibition of Notch signaling by the inhibitor DAPT rescued the impaired neuronal differentiation caused by CUL4B depletion.These results indicated that CUL4B deficiency caused abnormal activation of Notch pathway and hindered neuronal differentiation.In summary,we used the classical four-factor method to successfully induce wild-type and Cul4b knockout MEFs into miPSCs,which had the ability to differentiate into three germ layers in vivo and in vitro.CUL4B binds to the promoter of Cdh1 and exerts a transcriptional inhibitory function,thereby reducing the expression of E-Cadherin and hindering the process of reprogramming.Loss of CUL4B resulted in G1 phase,reduced proliferation and abnormally activated Notch signaling in miPSCs and the induced NPCs,thereby hindering the differentiation of NPCs into neurons.Part Ⅱ Construction and preliminary analysis of iPSCs from patients withCUL4B loss-of-function mutations and control individualsIn order to construct the iPSCs model from patients with CUL4B loss-of-function mutations,we analyzed two CUL4B mutation patients found in the previous study.The mutation of patient 1 was c.1564C>T,p.R388X,and the mutation of patient 2 was c.1007₁011del.,p.Ile336fs.By collecting somatic cells from the patients and the control individuals,reprogramming into hiPSCs,and performing characterization and functional analysis of hiPSCs,the following results were obtained:1.iPSCs from two patients with different mutations and two control individuals were established.Peripheral blood was collected from the two patients and one control individual,and mononuclear cells were isolated.Urine cells were obtained from the other control individual.Obtained hiPSCs of patients and control individuals were obtained by non-integrated plasmid transfection method.2.Characterization of hiPSCs.The characteristics of the obtained hiPSCs were analyzed.1)The expression of pluripotent genes was analyzed by immunofluorescence staining and RT-qPCR.The results showed that pluripotency protein markers OCT4,SOX2,SSEA4,TRA-1-60 and TRA-1-81 were highly expressed in hiPSCs.The expression levels of endogenous OCT4,SOX2 and NANOG reached the same level as embryonic stem cell H9;2)The three germ layers directional differentiation assay showed that hiPSCs from the patients and the control individuals could differentiate into the three germ layers;3)Exogenous gene expression detection.Genomic DNA was extracted from hiPSCs over 10 passages,and PCR amplification was performed with exogenous gene-specific primers.No exogenous gene expression was detected;4)Karyotype and STR locus analysis.Karyotype analysis found that no chromosomal aberrations occurred during the construction of hiPSCs.Genotyping of STR loci showed that hiPSCs were derived from the PBMCs of the corresponding patients,and there was no contamination from other cells;5)CUL4B mutation and expression analysis.Sequencing of CUL4B gene confirmed that the obtained hiPSCs from the two patients had the same mutations as the source cells.The hiPSCs from two patients has no CUL4B protein and significantly reduced CUL4B mRNA levels,indicating both CUL4B mutations cause mRNA degradation mediated by nonsense mutations.3.The hiPSCs were induced to differentiate into NPCs,and the expression of SOX2,NESTIN and PAX6 was detected by immunofluorescence to confirm the high purity of differentiated NPCs.4.The effect of CUL4B mutation on the proliferation of NPCs.EdU incorporation experiments showed that the proliferation ability of NPCs derived from patients with CUL4B mutations was significantly reduced.5.The effect of CUL4B mutation on NPCs migration.In order to analyze the effect of CUL4B mutation on NPCs migration,we conducted scratch wound experiment.The results showed that the migration ability of NPCs derived from patients with CUL4B mutations was significantly reduced.6.Detection of NPCs differentiation ability.NPCs were differentiated into neurons.Immunofluorescence staining showed that the differentiation of patient-derived NPCs into mature neurons was reduced,and the differentiation into GABA neurons was reduced.7.Transcriptome analysis:RNA-Seq analysis was performed on NPCs from the normal control and the patient.2075 differentially expressed genes were detected.Among them,31 up-regulated genes and 116 down-regulated genes exhibited more than 2-fold difference with Q-Value<0.01.1)GO-cfp analysis was done in the upregulated and downregulated genes.The biological classification of the two gene groups was roughly the same,while genes related to migration and proliferation were enriched in the down-regulated genes.Cluster heat map analysis and expression level verification of migration-related genes and proliferation-related genes were performed.Among the migration-related genes,the expression levels of SEMA6D,ONECUT2 and RHOD in patient-derived NPC were significantly decreased.Among the proliferation-related genes,the expression levels of TBX3,CTHRC1 and PRRX1 were significantly decreased.2)KEGG-Pathway enrichment analysis of the down-regulated genes showed that GABAergic synaptic related genes were enriched.Cluster heat map analysis and qPCR verification of these genes showed that GABRE,CACNA1A and PRCKB were significantly decreased in the patient-derived NPCs,consistent with the observed phenotype of CUL4B mutation leading to reduced GABA neuron differentiation.In summary,we successfully constructed hiPSCs cell lines from two patients with CUL4B mutations and differentiated them into NPCs.The derived NPCs exhibited slower proliferation,reduced migration and impaired differentiation into GABA neurons.Through RNA-sequencing analysis,we found genes related to proliferation,migration and GABA neuronal differentiation were down-regulated,suggesting that the phenotypes of NPCs from patients may be due to downregulation of these genes.The specific mechanism needs to be further analyzed.