Dimethyl sulfoxide stimulates the AhR-Jdp2 axis to control ROS accumulation in mouse embryonic fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-binding protein that responds to environmental aromatic hydrocarbons and stimulates the transcription of downstream phase I enzyme-related genes by binding the cis element of dioxin-responsive elements (DREs)/xenobiotic-responsive elements. Dimethyl sulfoxide (DMSO) is a well-known organic solvent that is often used to dissolve phase I reagents in toxicology and oxidative stress research experiments. In the current study, we discovered that 0.1% DMSO significantly induced the activation of the AhR promoter via DREs and produced reactive oxygen species, which induced apoptosis in mouse embryonic fibroblasts (MEFs). Moreover, Jun dimerization protein 2 (Jdp2) was found to be required for activation of the AhR promoter in response to DMSO. Coimmunoprecipitation and chromatin immunoprecipitation studies demonstrated that the phase I-dependent transcription factors, AhR and the AhR nuclear translocator, and phase II-dependent transcription factors such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) integrated into DRE sites together with Jdp2 to form an activation complex to increase AhR promoter activity in response to DMSO in MEFs. Our findings provide evidence for the functional role of Jdp2 in controlling the AhR gene via Nrf2 and provide insights into how Jdp2 contributes to the regulation of ROS production and the cell spreading and apoptosis produced by the ligand DMSO in MEFs.

Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128.

A cryptic microdeletion del(12)(p11.21p11.23) within an unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome.

In a patient diagnosed with both Kallmann syndrome (KS) and intellectual disability (ID), who carried an apparently balanced translocation t(7;12)(q22;q24)dn, array comparative genomic hybridization (aCGH) disclosed a cryptic heterozygous 4.7 Mb deletion del(12)(p11.21p11.23), unrelated to the translocation breakpoint. This novel discovery prompted us to consider the possibility that the combination of KS and neurological disorder in this patient could be attributed to gene(s) within this specific deletion at 12p11.21-12p11.23, rather than disrupted or dysregulated genes at the translocation breakpoints. To further support this hypothesis, we expanded our study by screening five candidate genes at both breakpoints of the chromosomal translocation in a cohort of 48 KS patients. However, no mutations were found, thus reinforcing our supposition. In order to delve deeper into the characterization of the 12p11.21-12p11.23 region, we enlisted six additional patients with small copy number variations (CNVs) and analyzed eight individuals carrying small CNVs in this region from the DECIPHER database. Our investigation utilized a combination of complementary approaches. Firstly, we conducted a comprehensive phenotypic-genotypic comparison of reported CNV cases. Additionally, we reviewed knockout animal models that exhibit phenotypic similarities to human conditions. Moreover, we analyzed reported variants in candidate genes and explored their association with corresponding phenotypes. Lastly, we examined the interacting genes associated with these phenotypes to gain further insights. As a result, we identified a dozen candidate genes: TSPAN11 as a potential KS candidate gene, TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B, and ETFBKMT as candidate genes for the neurodevelopmental disorder, and INTS13, REP15, PPFIBP1, and FAR2 as candidate genes for KS with ID. Notably, the high-level expression pattern of these genes in relevant human tissues further supported their candidacy. Based on our findings, we propose that dosage alterations of these candidate genes may contribute to sexual and/or cognitive impairments observed in patients with KS and/or ID. However, the confirmation of their causal roles necessitates further identification of point mutations in these candidate genes through next-generation sequencing.

A microdeletion del(12)(p11.21p11.23) with a cryptic unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome.

In an apparently balanced translocation t(7;12)(q22;q24)dn exhibiting both Kallmann syndrome (KS) and intellectual disability (ID), we detected a cryptic heterozygous 4.7 Mb del(12)(p11.21p11.23) unrelated to the translocation breakpoint. This new finding raised the possibility that KS combined with neurological disorder in this patient could be caused by gene(s) within this deletion at 12p11.21-12p11.23 instead of disrupted or dysregulated genes at the genomic breakpoints. Screening of five candidate genes at both breakpoints in 48 KS patients we recruited found no mutation, corroborating our supposition. To substantiate this hypothesis further, we recruited six additional subjects with small CNVs and analyzed eight individuals carrying small CNVs in this region from DECIPHER to dissect 12p11.21-12p11.23. We used multiple complementary approaches including a phenotypic-genotypic comparison of reported cases, a review of knockout animal models recapitulating the human phenotypes, and analyses of reported variants in the interacting genes with corresponding phenotypes. The results identified one potential KS candidate gene ( TSPAN11 ), seven candidate genes for the neurodevelopmental disorder ( TM7SF3 , STK38L , ARNTL2 , ERGIC2 , TMTC1 , DENND5B , and ETFBKMT ), and four candidate genes for KS with ID ( INTS13 , REP15 , PPFIBP1 , and FAR2 ). The high-level expression pattern in the relevant human tissues further suggested the candidacy of these genes. We propose that the dosage alterations of the candidate genes may contribute to sexual and/or cognitive impairment in patients with KS and/or ID. Further identification of point mutations through next generation sequencing will be necessary to confirm their causal roles.

TAA repeat variation in the GRIK2 gene does not influence age at onset in Huntington's disease.

Huntington's disease is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat whose length is the major determinant of age at onset but remaining variation appears to be due in part to the effect of genetic modifiers. GRIK2, which encodes GluR6, a mediator of excitatory neurotransmission in the brain, has been suggested in several studies to be a modifier gene based upon a 3' untranslated region TAA trinucleotide repeat polymorphism. Prior to investing in detailed studies of the functional impact of this polymorphism, we sought to confirm its effect on age at onset in a much larger dataset than in previous investigations. We genotyped the HD CAG repeat and the GRIK2 TAA repeat in DNA samples from 2,911 Huntington's disease subjects with known age at onset, and tested for a potential modifier effect of GRIK2 using a variety of statistical approaches. Unlike previous reports, we detected no evidence of an influence of the GRIK2 TAA repeat polymorphism on age at motor onset. Similarly, the GRIK2 polymorphism did not show significant modifier effect on psychiatric and cognitive age at onset in HD. Comprehensive analytical methods applied to a much larger sample than in previous studies do not support a role for GRIK2 as a genetic modifier of age at onset of clinical symptoms in Huntington's disease.

Disruption of neurexin 1 associated with autism spectrum disorder.

Autism is a neurodevelopmental disorder of complex etiology in which genetic factors play a major role. We have implicated the neurexin 1 (NRXN1) gene in two independent subjects who display an autism spectrum disorder (ASD) in association with a balanced chromosomal abnormality involving 2p16.3. In the first, with karyotype 46,XX,ins(16;2)(q22.1;p16.1p16.3)pat, NRXN1 is directly disrupted within intron 5. Importantly, the father possesses the same chromosomal abnormality in the absence of ASD, indicating that the interruption of alpha-NRXN1 is not fully penetrant and must interact with other factors to produce ASD. The breakpoint in the second subject, with 46,XY,t(1;2)(q31.3;p16.3)dn, occurs approximately 750 kb 5' to NRXN1 within a 2.6 Mb genomic segment that harbors no currently annotated genes. A scan of the NRXN1 coding sequence in a cohort of ASD subjects, relative to non-ASD controls, revealed that amino acid alterations in neurexin 1 are not present at high frequency in ASD. However, a number of rare sequence variants in the coding region, including two missense changes in conserved residues of the alpha-neurexin 1 leader sequence and of an epidermal growth factor (EGF)-like domain, respectively, suggest that even subtle changes in NRXN1 might contribute to susceptibility to ASD.

Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project.

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.

Application of Cancer Cell Reprogramming Technology to Human Cancer Research.

The cancer stem cell (CSC) hypothesis is an evolving concept of oncogenesis that has recently gained wide acceptance. By definition, CSCs exhibit continuous proliferation and self-renewal, and they have been proposed to play significant roles in oncogenesis, tumor growth, metastasis, chemoresistance, and cancer recurrence. The reprogramming of cancer cells using induced pluripotent stem cell (iPSC) technology is a potential strategy for the identification of CSC-related oncogenes and tumor-suppressor genes. This technology has some advantages for studying the interactions between CSC-related genes and the cancer microenvironment. This approach may also provide a useful platform for studying the mechanisms of CSCs underlying cancer initiation and progression. The present review summarizes the recent advances in cancer cell reprogramming using iPSC technology and discusses its potential clinical use and related drug screening.

Transcriptional regulation by zinc-finger proteins Sp1 and MAZ involves interactions with the same cis-elements.

Various transcription factors, such as Sp1 and MAZ, include C2H2-type zinc-finger motifs and are able to bind to GC-rich cis-elements that are distributed in the promoter regions of numerous mammalian genes. The consensus sequence of Sp1-binding sites is very similar to that of MAZ-binding sites. In fact, Sp1 and MAZ bind to the same cis-elements in the promoters of the genes for the receptor for serotonin 1A (HT1Ar), endothelial nitric-oxide synthase (eNOS), phenylethanolamine N-methyltransferase (PNMT), the receptor for parathyroid hormone (PTHr), MAZ and the major late promoter of adenovirus (AdMLP). It appears that two consecutive zinc-finger motifs of Sp1 and MAZ might be essential for the interaction of each protein with DNA. Sp1 and MAZ activated the expression of the genes for HT1Ar and PTHr, as well as AdMLP. Both Sp1 and MAZ inhibited the expression of the gene for MAZ, while expression of the gene for eNOS was enhanced by Sp1 and repressed by MAZ. These observations suggest that both Sp1 and MAZ might have dual functions in the regulation of gene expression. Our results suggest, furthermore, that histone deacetylases are involved in autorepression of the gene for MAZ, while expression of DNA methyltransferase I is associated with suppression of the expression of the gene for MAZ by Sp1. Thus, both deacetylation and methylation might be involved in the regulation of expression of individual genes, with different zinc-finger proteins binding to the same cis-elements but recruiting different proteins, such as methylases and acetylases, to the transcriptional complex.

Genetic materials at the gene engineering division, RIKEN BioResource Center.

Genetic materials are one of the most important and fundamental research resources for studying biological phenomena. Scientific need for genetic materials has been increasing and will never cease. Ever since it was established as RIKEN DNA Bank in 1987, the Gene Engineering Division of RIKEN BioResource Center (BRC) has been engaged in the collection, maintenance, storage, propagation, quality control, and distribution of genetic resources developed mainly by the Japanese research community. When RIKEN BRC was inaugurated in 2001, RIKEN DNA Bank was incorporated as one of its six Divisions, the Gene Engineering Division. The Gene Engineering Division was selected as a core facility for the genetic resources of mammalian and microbe origin by the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan in 2002. With support from the scientific community, the Division now holds over 3 million clones of genetic materials for distribution. The genetic resources include cloned DNAs, gene libraries (e.g., cDNA and genomic DNA cloned into phage, cosmid, BAC, phosmid, and YAC), vectors, hosts, recombinant viruses, and ordered library sets derived from animal cells, including human and mouse cells, microorganisms, and viruses. Recently genetic materials produced by a few MEXT national research projects were transferred to the Gene Engineering Division for further dissemination. The Gene Engineering Division performs rigorous quality control of reproducibility, restriction enzyme mapping and nucleotide sequences of clones to ensure the reproducibility of in vivo and in vitro experiments. Users can easily access our genetic materials through the internet and obtain the DNA resources for a minimal fee. Not only the materials, but also information of features and technology related to the materials are provided via the web site of RIKEN BRC. Training courses are also given to transfer the technology for handling viral vectors. RIKEN BRC supports scientists around the world in the use of valuable genetic materials.

Candidate loci for Zimmermann-Laband syndrome at 3p14.3.

A male with 46,XY,t(3;17)(p14.3;q24.3) presented with gingival hyperplasia, hypertrichosis, unusually large ears and marked hypertrophy of the nose, characteristic of the Zimmermann-Laband syndrome (ZLS). Other features include large facial bones and mandibles, large protruding upper lip, enlarged fingers and toes, strabismus, and enlarged phallus. Knowledge of a 46,XX,t(3;8)(p21.2;q24.3) reported previously in a mother and daughter with ZLS suggests that the 3p14.3-p21.2 region may contain a gene responsible for ZLS. We have reassessed the chromosome 3 breakpoint region of the t(3;8) and revised its breakpoint location to 3p14.3, based upon an updated human genome sequence assembly. Using fluorescence in situ hybridization (FISH) with BAC clones, we have also identified a breakpoint spanning clone at 3p14.3 in our t(3;17) patient, thereby narrowing the breakpoint to a region of approximately 200 kb. These data suggest that the gene responsible for ZLS is located in 3p14.3 and implicates four likely candidate genes in this region: CACNA2D3, encoding a voltage-dependent calcium channel, LRTM1, a gene of unknown function embedded within CACNA2D3, WNT5A, encoding a secreted signaling protein of the WNT family, and ERC2, which codes for a synapse protein.

Brain-derived neurotrophic factor does not influence age at neurologic onset of Huntington's disease.

In Huntington's disease (HD), genetic factors in addition to the HD CAG repeat mutation play a significant role in determining age at neurologic onset. Brain-derived neurotrophic factor (BDNF), a survival factor for striatal neurons, has been implicated as a target of regulation by huntingtin and is an attractive candidate as a genetic modifier. We tested this hypothesis by genotyping a SNP known to alter BDNF function (rs6265, also termed Val66Met) and a SNP associated with Alzheimer disease (BDNF C270T), along with two BDNF intronic SNPs (rs7103411, rs11030104), in 228 cases with extreme young onset and 329 cases with extreme old onset of HD. No differences were seen between groups for allele frequencies or genotype frequencies for any SNP. Furthermore, no association to onset age was seen in GEE models controlling for HD repeat size or in haplotype analyses of these SNPs. These results indicate that BDNF does not influence significantly the mechanisms in HD pathogenesis that lead to neurologic onset.

Roles of histone acetylation in the Dnmt1 gene expression.

The DNA methylation plays a key role in the regulation of gene expression, genomic imprinting and X chromosome inactivation and has been shown to be essential for mammalian development. The Dnmt1 is one of the DNA methyltransferases that catalyzed DNA methylation on CpG dinucleotides. The Dnmt1 is constitutively expressed and is required for the maintenance of global methylation after DNA replication. In this study, we investigated the effects of histone deacetylase (HDAC) inhibitor and DNA demethylation agent on promoter activity of mouse Dnmt1 gene in somatic cells. The promoter activity of Dnmt1 gene was increased approximately 2-fold in the treatment of cells by Trichostatin A (TSA) at 1 x 10(-8) M, as compared with that without of treatment of TSA. By contrast, treatment with 5-azacytidine (5aza-C) did not affect the promoter activity of the Dnmt1 gene. These results indicate that the Dnmt1 gene is possibly to regulated by histone acetylation.

Control elements of Dnmt1 gene are regulated in cell-cycle dependent manner.

The Dnmt1 gene is constitutively expressed and is required for the maintenance of global methylation after DNA replication. We investigated here the effects of histone deacetylase (HDAC) inhibitor and DNA demetylation agent on promoter activity of mouse Dnmt1 gene in somatic cells. The promoter activity of Dnmt1 gene was increased approximately 2-fold in the treatment of cells by Tricostatin A (TSA) at 1 x 10(-8) M, as compared with that without treatment of TSA. By contrast, treatment with 5-azacytidne (5aza-C) did not affect the promoter activity of the Dnmt1 gene. This result indicates the Dnmt1 gene is possibly regulated by histone acetylation. We also examined the expression levels of Dnmt1 gene and of its control elements like Sp1, Sp3 and p300 by the chromatin immunoprecipitation and Western blot analysis. The expression of Dnmt1 gene is observed at early S phase. Sp1 is recruited mainly at the G1 phase and Sp3 is recruited at the early S phase. p300 is also obviously recruited at the second S phase. These data indicated that the regulators of Dnmt1 gene were controlled in cell-cycle dependent manner.

Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteins.

The Sp family is a family of transcription factors that bind to cis-elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis-element (nucleotides -161 to -147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis-element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3.