Systematic identification and characterization of regulatory elements derived from human endogenous retroviruses.

Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)-type retrotransposons (HERV/LTRs) have regulatory elements that possibly influence the transcription of host genes. We systematically identified and characterized these regulatory elements based on publicly available datasets of ChIP-Seq of 97 transcription factors (TFs) provided by ENCODE and Roadmap Epigenomics projects. We determined transcription factor-binding sites (TFBSs) using the ChIP-Seq datasets and identified TFBSs observed on HERV/LTR sequences (HERV-TFBSs). Overall, 794,972 HERV-TFBSs were identified. Subsequently, we identified "HERV/LTR-shared regulatory element (HSRE)," defined as a TF-binding motif in HERV-TFBSs, shared within a substantial fraction of a HERV/LTR type. HSREs could be an indication that the regulatory elements of HERV/LTRs are present before their insertions. We identified 2,201 HSREs, comprising specific associations of 354 HERV/LTRs and 84 TFs. Clustering analysis showed that HERV/LTRs can be grouped according to the TF binding patterns; HERV/LTR groups bounded to pluripotent TFs (e.g., SOX2, POU5F1, and NANOG), embryonic endoderm/mesendoderm TFs (e.g., GATA4/6, SOX17, and FOXA1/2), hematopoietic TFs (e.g., SPI1 (PU1), GATA1/2, and TAL1), and CTCF were identified. Regulatory elements of HERV/LTRs tended to locate nearby and/or interact three-dimensionally with the genes involved in immune responses, indicating that the regulatory elements play an important role in controlling the immune regulatory network. Further, we demonstrated subgroup-specific TF binding within LTR7, LTR5B, and LTR5_Hs, indicating that gains or losses of the regulatory elements occurred during genomic invasions of the HERV/LTRs. Finally, we constructed dbHERV-REs, an interactive database of HERV/LTR regulatory elements (http://herv-tfbs.com/). This study provides fundamental information in understanding the impact of HERV/LTRs on host transcription, and offers insights into the transcriptional modulation systems of HERV/LTRs and ancestral HERVs.

Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus.

Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging "allele-specific" functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage.

Rapid and cost-effective high-throughput sequencing for identification of germline mutations of BRCA1 and BRCA2.

Genetic testing for breast cancer predisposing genes, BRCA1 and BRCA2, can take advantage for early identification of carriers with pathogenic germline mutations. However, conventional approaches based on Sanger sequencing are laborious and expensive. Next-generation sequencing technology has a great impact on investigation of medical genomics and now applied clinical genetics. We provide a protocol based on a pool and capture method followed by high-throughput sequencing, which realizes a rapid, high-quality, high-accuracy and low-cost testing for mutations in BRCA1 and BRCA2 by using small amounts of input DNA. Custom capture probes were designed for 195 kb regions encompassing the entire BRCA1 and BRCA2. DNA libraries of 96 samples with distinct indices were pooled before hybridizing to the capture probes, which largely reduced labor and cost. The captured library was run on the Illumina MiSeq sequencer. We applied the method to 384 Japanese individuals including 11 patients with breast cancer whose mutation statuses had been determined by standard clinical testing and 373 individuals from a general population. 99.99% of coding exons and their 20 bp flanking regions were covered with a minimum of 20 reads and the average depth was 179.5, supporting confident variant detection. The sequencing method rendered concordant results for 11 patients with breast cancer compared with the standard clinical testing including nine mutations in eight patients. Among 373 individuals from the general population, novel stop gain and frameshift deletion in BRCA2 were identified, which led to truncated protein and were most likely to be pathogenic. The result suggests the importance of a large-scale population-wide screening for carriers of mutations in these genes.

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway.

We investigated the oncogenic role of SETDB1, focusing on non-small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT-ß-catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1.

BRCT domain of DNA polymerase µ has DNA-binding activity and promotes the DNA polymerization activity.

DNA polymerase µ (pol µ) catalyzes nonhomologous end-joining in DNA double-stranded break repair. Pol µ consists of an amino-terminal BRCA1 carboxyl-terminal homology (BRCT) domain and a pol ß-like region, which contains the catalytic site. By DNA cellulose column chromatography, using full-length pol µ and five different deletion mutants, we found that the amino-terminal region has double-stranded DNA (dsDNA)-binding activity. Pol µ without BRCT domain reduces the DNA polymerization activity when compared to full-length pol µ. Observation by atomic force microscopy showed that full-length pol µ binds to the ends and middle part of dsDNA. Pol µ lacking the amino-terminal region or with a mutation within the BRCT domain bound only to DNA ends, whereas the amino-terminal region with the BRCT domain bound to both the ends and the middle part of dsDNA (mpdDNA). Terminal deoxynucleotidyltransferase, which, like pol µ, belongs to the X family DNA polymerases, also bound to mpdDNA through its amino-terminal region.

TdIF2 is a nucleolar protein that promotes rRNA gene promoter activity.

Terminal deoxynucleotidyltransferase (TdT) interacting factor 2 (TdIF2) is an acidic protein that binds to TdT. TdIF2 binds to DNA and core histones and contains an acidic-amino acid-rich region in its C-terminus. It has therefore been suggested to function as a histone chaperone within the nucleus. TdIF2 localized within the nucleolus in HEK 293T cells, and its N-terminal (residues 1-234) and C-terminal (residues 606-756) regions were crucial for the nucleolar localization. A chromatin immunoprecipitation (ChIP) assay showed that TdIF2 associated with the promoter of human ribosomal RNA genes (hrDNAP), and an in vitro luciferase assay system showed that it promoted hrDNAP activity. Using the yeast two-hybrid system with TdIF2 as the bait, we isolated the cDNA encoding HIV Tat interactive protein 60 (Tip60), which has histone acetyltransferase (HAT) activity, as a TdIF2-binding protein. TdIF2 bound to Tip60 in vitro and in vivo, inhibited the Tip60 HAT activity in vitro and co-localized with Tip60 within the nucleolus. In addition, TdIF2 promotes upstream binding factor (UBF) acetylation in vivo. Thus, TdIF2 might promote hrDNAP activity by suppressing Tip60's HAT activity and promoting UBF acetylation.

Symbiotic bacteria-dependent expansion of MR1-reactive T cells causes autoimmunity in the absence of Bcl11b.

MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αß T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential.

Early manifestations and differential gene expression associated with photoreceptor degeneration in Prom1-deficient retina.

Retinitis pigmentosa (RP) and macular dystrophy (MD) are characterized by gradual photoreceptor death in the retina and are often associated with genetic mutations, including those in the prominin-1 (Prom1) gene. Prom1-knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and constriction of retinal blood vessels. The mechanisms underlying such degeneration have remained unclear, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. Whereas gene expression was not affected at 2 weeks, the expression of several genes was altered at 3 weeks in the Prom1-KO retina, with the expression of that for endothelin-2 (Edn2) being markedly upregulated. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice reared in the dark. Treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis and retinal vessel stenosis in Prom1-KO mice. Our findings thus reveal early manifestations of retinal degeneration in a model of RP/MD and suggest potential therapeutic agents for these diseases. This article has an associated First Person interview with the first author of the paper.

TdT interacting factor 1 enhances TdT ubiquitylation through recruitment of BPOZ-2 into nucleus from cytoplasm.

We isolated human cDNA clone encoding Bood POZ containing gene type 2 (BPOZ-2) as a gene with a product that binds to TdT interacting factor 1 (TdIF1) using a yeast two-hybrid system. BPOZ-2 is an adaptor for E3 ligase CUL3 and participates in developmental processes. The binding between BPOZ-2 and TdIF1 was confirmed by GST pull-down and immunoprecipitation assays using specific antibodies against BPOZ-2 and TdIF1 in vitro and in vivo. Although when BPOZ-2 solely was expressed in COS7 cells, BPOZ-2 was observed mainly within the cytoplasm, co-transfection of pEGFP-BPOZ-2 and pDsRed-TdIF1 into COS7 cells resulted in co-localization of EGFP-BPOZ-2 and DsRed-TdIF1 within the nucleus. TdIF1 may recruit BPOZ-2 into the nucleus from the cytoplasm by directly binding to BPOZ-2. BPOZ-2 enhanced TdT ubiquitylation when TdIF1 was expressed together with BPOZ-2 in 293T cells, strongly suggesting that the recruitment of BPOZ-2 into the nucleus from the cytoplasm is significant for the TdT ubiquitylation within the nucleus.

BPOZ-2 directly binds to eEF1A1 to promote eEF1A1 ubiquitylation and degradation and prevent translation.

Bood POZ containing gene type 2 (BPOZ-2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ-2 binding protein by screening a human thymus cDNA library using a yeast two-hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome-dependent degradation of misfolded or unfolded proteins. The binding between BPOZ-2 and eEF1A1 was confirmed by pull-down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ-2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ-2 and Domains I and III in eEF1A1. BPOZ-2 and eEF1A1 over-expressed in HEK 293T cells co-localized as speckles within the cytoplasm. BPOZ-2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ-2. BPOZ-2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.

Bood POZ containing gene type 2 is a human counterpart of yeast Btb3p and promotes the degradation of terminal deoxynucleotidyltransferase.

Bood POZ containing gene type 2 (BPOZ-2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2 bound to E3 ligase CUL3 in vitro and in vivo. BPOZ-2 itself was ubiquitinated through the CUL3-based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ-2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ-2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ-2-binding protein using a yeast two-hybrid system by screening a human thymus cDNA library. TdT, BPOZ-2, and CUL3 formed a ternary complex in vivo. TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co-expression of BPOZ-2 and CUL3 or BPOZ-2 in 293T cells, respectively.

Direct binding of ligandin to uridine 5'-diphosphate glucuronosyltransferase 1A1.

AIM: Bilirubin, a final degradation product of heme produced mainly in the spleen, is carried to the liver through its binding to albumin in the blood circulation. After its transport to hepatocytes, ligandin (glutathione S-transferase; GST) carries bilirubin to the endoplasmic reticulum (ER). uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) glucuronidates bilirubin for solubilization in the ER. METHODS: By GST pull-down and co-immunoprecipitation assays, GSTA2, a member of the alpha-class of GST, was observed to directly bind to UGT1A1 through the region present inside the ER. RESULTS: GSTA2 was detected in the microsomal fraction together with the cytosolic fraction after hepatocyte fractionation. CONCLUSION: These results strongly suggest that bilirubin is directly delivered to UGT1A1 from ligandin for glucuronidation.

Exome and copy number variation analyses of Mayer-Rokitansky-Küster- Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital absence of the vagina and uterus. We conducted genome-wide SNP analyses and exome sequencing to detect the causes of MRKH syndrome. We identified de novo variants of MYCBP2, NAV3, and PTPN3 in three families and a variant of MYCBP2 in a sporadic case. Here, we demonstrated the partial genetic makeup of Japanese MRKH syndrome.

Long non-coding RNA p10247, high expressed in breast cancer (lncRNA-BCHE), is correlated with metastasis.

Recent studies have shown that long non-coding RNAs (lncRNAs) have key functions during breast cancer development. Considering the complexity of IncRNAs regulatory network, the identification of novel and functional lncRNAs associated with breast cancer is thus very important. By using Agilent LncRNA Human Gene Expression Microarray, we identified a number of lncRNAs that were differentially expressed in breast cancer compared to their corresponding adjacent tissues. According to the microarray, the expression of p10247, henceforth named as lncRNA-BCHE (standing for lncRNA high expressed in breast cancer), was found to be uniformly higher in all the five breast cancer tissues tested, and this was further confirmed in 56 breast cancer tissues by real-time RT-PCR. The function of lncRNA-BCHE in breast cancer cells was tested by knockdown and over-expression experiments in vitro. We also analyzed the public cohorts of breast cancer patients on the Kaplan Meier plotter platform. Clinical analysis revealed that the expression of lncRNA-BCHE was significantly correlated with advanced clinical stage and lymph node metastasis. Our data indicate that lncRNA-BCHE regulates the growth, migration and invasion of breast cancer cells. In addition, we found that these functions are mediated, at least in part, by the regulation of integrin subunit beta 1 (ITGB1) levels. The expression of ITGB1 serves as a negative prognostic factor and metastasis risk predictor in breast cancer, irrespective of subtype and therapeutic regimen. In summary, our results suggest that lncRNA-BCHE is an oncogenic lncRNA enhancing the growth and metastatic potential of breast cancer cells, and a potential predictor of breast cancer metastatic progression.

Germline Variants of Prostate Cancer in Japanese Families.

Prostate cancer (PC) is the second most common cancer in men. Family history is the major risk factor for PC. Only two susceptibility genes were identified in PC, BRCA2 and HOXB13. A comprehensive search of germline variants for patients with PC has not been reported in Japanese families. In this study, we conducted exome sequencing followed by Sanger sequencing to explore responsible germline variants in 140 Japanese patients with PC from 66 families. In addition to known susceptibility genes, BRCA2 and HOXB13, we identified TRRAP variants in a mutually exclusive manner in seven large PC families (three or four patients per family). We also found shared variants of BRCA2, HOXB13, and TRRAP from 59 additional small PC families (two patients per family). We identified two deleterious HOXB13 variants (F127C and G132E). Further exploration of the shared variants in rest of the families revealed deleterious variants of the so-called cancer genes (ATP1A1, BRIP1, FANCA, FGFR3, FLT3, HOXD11, MUTYH, PDGFRA, SMARCA4, and TCF3). The germline variant profile provides a new insight to clarify the genetic etiology and heterogeneity of PC among Japanese men.

Identification of novel exonic mobile element insertions in epithelial ovarian cancers.

Mobile elements comprise about half of the human genome. Three active mobile element families (L1, Alu, and SVA) possibly cause diseases such as cancer. We conducted mobile element insertion (MEI) profiling of 44 epithelial ovarian cancers using exome-sequencing data. We identified a total of 106 MEIs using the Mobster program, 8 of which were novel exonic MEIs.

Molecular characterization of an intact p53 pathway subtype in high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.

SOX7 is down-regulated in lung cancer.

BACKGROUND: SOX7 is a transcription factor belonging to the SOX family. Its role in lung cancer is unknown. METHODS: In this study, whole genomic copy number analysis was performed on a series of non-small cell lung cancer (NSCLC) cell lines and samples from individuals with epidermal growth factor receptor (EGFR) mutations using a SNP-Chip platform. SOX7 was measured in NSCLC samples and cell lines, and forced expressed in one of these lines. RESULTS: A notable surprise was that the numerous copy number (CN) changes observed in samples of Asian, non-smoking EGFR mutant NSCLC were nearly the same as those CN alterations seen in a large collection of NSCLC from The Cancer Genome Atlas which is presumably composed of predominantly Caucasians who often smoked. However, four regions had CN changes fairly unique to the Asian EGFR mutant group. We also examined CN changes in NSCLC lines. The SOX7 gene was homozygously deleted in one (HCC2935) of 10 NSCLC cell lines and heterozygously deleted in two other NSCLC lines. Expression of SOX7 was significantly downregulated in NSCLC cell lines (8/10, 80%) and a large collection of NSCLC samples compared to matched normal lung (57/62, 92%, p= 0.0006). Forced-expression of SOX7 in NSCLC cell lines markedly reduced their cell growth and enhanced their apoptosis. CONCLUSION: These data suggest that SOX7 is a novel tumor suppressor gene silenced in the majority of NSCLC samples.

Ubiquitylation of terminal deoxynucleotidyltransferase inhibits its activity.

Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.