Identification And Characterization Of The Gene Responsible For A Novel X-Linked Mental Retardation Syndrome

Mental retardation(MR) is characterized by sub-average cognitive functioning and deficits in social and adaptive skills,with onset under the age of 18.Because MR has a high prevalence and many MR patients need special care,it represents a major challenge and a high burden for society and families.Because of the lack of effective medical treatment for individuals with MR,identification of the etiological factors is critical for developing appropriate stragegies in the prevention,intervention and treatment of MR.X-linked genetic defects have been considered to be one of the important causes of mental retardation.X-linked mental retardation(XLMR) conditions are subdivided into three classes based on their clinical presentations:syndromic (S-XLMR,MRXS),neuromuscular disorders and non-syndromic(NS-XLMR,MRX) forms.At present,there are more than 170 distinct MR loci mapped to the X chromosome and 82 genes have been cloned.We identified a family with XLMR during a population survey for common hereditary diseases in Zoucheng,Shandong Province,China.Because the clinical features in this family were similar with that of Smith-Fineman-Myers syndrome (SFMS),this family was initially reported as having putative SFMS.The causative gene was mapped by linkage analysis to an interval of 10.18 Mb on Xq25.5.However, SFMS was later found to be caused by mutations in XNP,a gene located at Xq13. This suggested that either SFMS has genetic heterogeneity or this Zoucheng XLMR family represents a new syndrome that is distinct from SFMS in genetic etiology and in unrecognized phenotypes.To understand the molecular mechanisms of this disease, we identified the causative gene of Zoucheng family with XLMR and characterized its expression pattern,transcriptional regulation,subcellular localization and functional importance in cell proliferation.PART ONE Loss of Function Mutation in CUL4B Gene Causes a New X-linked Mental Retardation Syndrome:MRXS15The facts that the causative gene of the disease in the Zoucheng family with XLMR was mapped to Xq25 and the SFMS gene XNP was excluded as the cause of the disease suggest that cases in this family with XLMR probably represent a syndrome different from SFMS.Therefore,we revisited the family and reexamined the patients in 2005.There are 7 affected males in 6 sibships and no female is affected in this family. All adult patients showed mild to moderate MR and led their daily lives under the care and supervision of their parents and other relatives.None of the adult patients were able to speak a single word during their lifetimes.In addition to MR,several other physical features were also observed in patients,including short stature, brachydactyly,large tongue(macroglossia),and a unique gait of walking with the toes pointing inward.Neuroimaging by magnetic resonance imaging and computed axial tomography scanning revealed no structural abnormalities.Peripheral blood tests revealed that monocyte counts were remarkably increased,whereas the total WBC counts were in the normal range.To identify the gene responsible for this family we performed mutation analysis of candidate genes in the candidate region.After excluding the first 9 genes,we found a nonsense mutation in the 10th gene tested,CUL4B.A base substitution at 1564 in exon 9,c.1564C→T,converted a codon for arginine(CGA) to a termination codon (TGA),p.R388X.Further studies showed that this mutation cosegregated with the disease phenotype in the family.There was no 1564T allele detected among 312 X chromosomes from 264 unrelated,unaffected Chinese controls.Thus,it is unlikely that this mutation represents a polymorphism.We next measured the mRNA levels of CUL4B in peripheral leukocytes of patients,carriers,and controls by real time PCR.We found only～30%of the mRNA,a 70%reduction,was retained in the patients when compared with the controls.These results suggest that the mRNA of CUL4B was readily eliminated in those patients by the non-sense mediated decay(NMD) mechanism and the loss of function mutation in CUL4B was the cause of MRXS15.Interestingly,the CUL4B mRNA levels in carriers were as high as those in unaffected individuals.This was probably achieved by a selection against cells expressing the 1564T allele.To test this hypothesis,we evaluated the X-chromosome inactivation pattern in carriers and unaffected females.We found that X-chromosome inactivation was extremely skewed in all the informative carriers tested and only the wild-type allele was detected in leukocyte cDNA from those carriers.With the identification of the CUL4B as the causative gene and the presence of some unique clinical features,we proposed that the disease in this family represents a novel syndrome(MRXS15).These results,together with the findings of mutations of CUL4B in 8 families by Stratton et al,indicate that CUL4B is critical in cognition as well as other aspects of human development.PART TWO Expression Pattern of CUL4 and Transcriptional Regulatory Mechanism of CUL4BAlthough genetic studies showed that CUL4B plays an important role in development,little is known about the molecular function of CUL4B.CUL4 has two closely related paralogs,CUL4A and CUL4B,in mammals.Although CUL4A and CUL4B protein are 83%identical,recent studies have shown these two genes may have different functions.Therefore,in this section we investigated the expression pattern of CUL4 and characterized the transcriptional regulation of CUL4B expression by using the luciferase reporter assay.1.Real time PCR analysis revealed that CUL4B is expressed in all tissues detected,with the highest levels in brain and spleen,moderate in lung,liver,kidney and thymus,and lowest levels in skeletal muscle,nearly 10-fold less than that in brain. CUL4A is also ubiquitously expressed.But differently from CUL4B,there is no significant difference in the expression levels among different tissues.2.We performed Western blotting analysis of a set of 20 human cell lines. While CUL4B protein is expressed in all cell lines examined,its expression level is varied.Similar to the mRNA expression pattern in fetal human tissues,CUL4B protein is high in nervous system cell lines,but is low in all hematopoietic cell lines examined.In contrast,CUL4A is expressed at similar levels in these cell lines.3.Computer-based analysis of the 1kb region upstream of the translational start site of human CUL4B gene revealed a putative TATA box,～50bp upstream of ATG. In addition,some other potential cis-regulatory elements such as CAAT box,GATA box,Sp1 and E2F were identified in this region.4.P-CULB-952 showed a greater promoter activity in different cell lines including HeLa,LO2,U87 and SH-SY5Y than the promoterless luciferase vector, pGL3-basic,suggesting that the region～952bp upstream of ATG contains functional elements for the transcription of human CUL4B in different tissues.To further identify the sequence that is necessary for basal transcription of human CUL4B in different cell lines,we analyzed by reporter gcne assays various 5' deletions of the promoter. The promoter activity was drastically decreased in constructs with deletions between positions -476 and -452 in U87 and SH-SY5Y cells,but not in HeLa,HEK293 and LO2 cells,suggesting that the region from -476bp to -452bp contains nerve-specific cis-elements that arc crucial for the basal transcription of the CUL4B gene in nervous tissue.5.Using mutagenesis analysis,we found that only the mutation of ELK1 site located in the region between -476 and -452 resulted in significantly reduced promoter activity in SH-SY5Y cells.However,this reduction of promoter activity was not observed in C6 and U87 cells,indicating that there is a difference in transcriptional regulation of CUL4B gene between neuronal cells and glial cells and ELK1 element is the neuronal specific cis-element essential for the CUL4B transcription.PART THREE Subcellular Localization of CUL4 and Identification of a Functional NLS of CUL4BTo further understand the function of CUL4B,we investigated the subcellular localization of CUL4A and CUL4B and identified the functional NLS of CUL4B.1.To investigate the subcellular localization of CUL4A and CUL4B,we first fused CUL4A and CUL4B cDNA containing the complete open reading frame to C-terminal end of EGFP to construct pEGFP-C1-CUL4A and pEGFP-C1-CUL4B expression vector and transiently transfected into different cell lines.The results showed that the subcellular localization of EGFP-CUL4A varies among different cell lines,while EGFP-CUL4B was exclusively localized in the nucleus in all cell lines examined.Therefore,despite their strong sequence homology,CUL4B and CUL4A have different patterns of subcellular distribution in transfected cells.2.We next examined the subcellular localization of CUL4B-Myc-His fusion protein in which the tagged proteins Myc and His are at the C-terminal end of CUL4B by immunofluorescence using anti-Myc antibody.The results showed that like EGFP-CUL4B,CUL4B-Myc-His mainly distributed in nucleus in different cell lines.3.We found that lacking of C-terminus did not impair the nuclear accumulation of CUL4B.In contrast,CUL4B with N-terminal end truncated was unable to accumulate in the nucleus and was exclusively observed in the cytoplasm.The N-terminal fragment of CUL4B was able to guide EGFP to the nucleus.These results indicated that N-terminus but not C-terminal region of CUL4B regulates its nuclear import.4.We used web-based computer program PSORTâ…¡to predict the functional NLS in the amino acid sequence of CUL4B.According to calculations by this program,four sequences were suggested as putative NLSs of CUL4B,three in the first 100 residues and the other in the C-terminus.To determine which of the three candidate NLSs in the N-terminus are actually responsible for nuclear import of CUL4B,a series truncation mutant constructs were generated and transiently expressed in HeLa cells.The result showed that ³⁷KKRK⁴⁰ is the functional NLS of CUL4B.Using mutation analysis,we found 38K and 39R were the key amino acids regulating the nuclear localization of CUL4B.5.To further understand the mechanism of nuclear import of CUL4B,we performed in vitro GST pulldown experiments to investigate the ability of CUL4B to interact with importinαproteins.The results showed that all importinαs(importinα1,α3,α5) tested in this assay could interact with CUL4B in vitro in a NLS-mediated way,suggesting that these importinαs are all capable of transporting CUL4B into the nucleus in conjunction with importinβ.PART FOUR he Role of CUL4B in Cell Proliferation and Cell Cycle RegulationThe findings that X-chromosome inactivation was extremely skewed in carriers and only the wild-type allele was detected in leukocyte cDNA from those carriers suggest that cells lacking CUL4B are impaired in proliferation and strongly selected against in vivo.In addition,previous studies have shown that CUL4A is involved in regulation of several key cell cycle regulators.These results prompted us to examine whether CUL4B also plays an important role in cell proliferation and cell cycle regulation.1.We first evaluated the effects of CUL4B overexpression on cell proliferation by MTT assay.We found that upregulationof CUL4B expression can promote growth rate of HEK293 cells2.Next,we employed CUL4B-specific RNAi to stably knock down endogenous CUL4B expression in HeLa cells and determined changes in cell proliferation.The results of MTT assay indicated that CUL4B depletion led to a significantly decreased growth rate of HeLa cells.Using TUNEL assay,we evaluated the effect of CUL4B down-regulation on cell apoptosis,but no difference was observed.These results suggest that the inhibition of cell growth may be mediated by cell cycle arrest.To test our hypothesis,BrdU incorporation assay and FACS analysis were employed.Consistent with MTT assay,down-regulation of CUL4B showed a significant decrease in the number of BrdU positive cells compared to the control group.Interestingly,Cell cycle distributions by FACS analysis showed the amount of cells in S phase increased about 10%in CUL4B depletion cells.Taken together,these results indicate that the inhibition of proliferation caused by constitutive silencing of CUL4B was mediated by S phase arrest.3.To understand the molecular basis for CUL4B depletion-induced cell cycle arrest in HeLa cells,we examined the expression levels of several cell cycle regulators by Western blotting analysis.Of the proteins examined,silencing of CUL4B only led to the selective accumulation of cyclin E as compared to control cells.4.Since CUL4B was mainly localized in nuclei,we further examined whether nuclear localization of CUL4B was required for its functions in regulating of cell proliferation.We found that an over-expression of full-length CUL4B and CUL4A significantly increased the proliferation of cells compared with untransfected cells.In contrast,expression of constructs in which the NLS or N-terminal region of CUL4B had been deleted was unable to promote cell proliferation.Rescue experiments also showed that wildtype CUL4B could restore DNA synthesis and relieve the accumulation of cyclin E protein in CUL4B-depleted HeLa cells.These data indicated that the nuclear localization of CUL4B was required for its regulation of cell proliferation.Taken together,this study provided the first evidence of CUL4B mutation as the cause of a human disease.In addition,we analyzed the expression pattern,subcellular localization,transcriptional regulation of CUL4B and its role in cell proliferation. These results advanced our understanding of the etiology of XLMR and the functions of CUL4B in human development.