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PubMed2002

CTCF maintains differential methylation at the Igf2/H19 locus.

Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles. Although the essential DNA methyltransferases have been discovered, proteins that regulate the sequence-specific establishment and maintenance of allelic methylation have not been identified. One candidate regulator of methylation, the zinc-finger protein CTCF, binds to the imprinting control region (ICR) of the genes Igf2 (encoding insulin-like growth factor 2) and H19 (fetal liver mRNA; refs. 1,2). The unmethylated maternal ICR is a chromatin boundary that prevents distant enhancers from activating Igf2 (refs. 3-6). In vitro experiments have suggested that CTCF mediates boundary activity of the maternal ICR, and that methylation of the paternal ICR abolishes this activity by preventing CTCF binding. Using mice with point mutations in all four CTCF sites in the ICR, we show that maternally transmitted mutant ICRs in neonatal mice acquire a substantial but heterogeneous degree of methylation. Mutant ICRs in oocytes and blastocysts are not methylated, however, indicating that binding of CTCF is not required to establish the unmethylated ICR during oogenesis. We also show that the mutant ICR lacks enhancer-blocking activity, as the expression of Igf2 is activated on mutant maternal chromosomes. Conversely, maternal H19 expression is reduced, suggesting a positive role for CTCF in the transcription of that gene. This study constitutes the first in vivo demonstration of the multiple functions of CTCF in an ICR.

Alleles↗
PubMed2004

Silencing of imprinted CDKN1C gene expression is associated with loss of CpG and histone H3 lysine 9 methylation at DMR-LIT1 in esophageal cancer.

The putative tumor suppressor CDKN1C is an imprinted gene at 11p15.5, a well-known imprinted region often deleted in tumors. The absence of somatic mutations and the frequent diminished expression in tumors would suggest that CDKN1C expression is regulated epigenetically. It has been, however, controversial whether the diminution is caused by imprinting disruption of the CDKN1C/LIT1 domain or by promoter hypermethylation of CDKN1C itself. To clarify this, we investigated the CpG methylation index of the CDKN1C promoter and the differentially methylated region of the LIT1 CpG island (differentially methylated region (DMR)-LIT1), an imprinting control region of the domain, and CDKN1C expression in esophageal cancer cell lines. CDKN1C expression was diminished in 10 of 17 lines and statistically correlated with the loss of methylation at DMR-LIT1 in all but three. However, there was no statistical correlation between CDKN1C promoter MI and CDKN1C expression. Furthermore, loss of CpG methylation was associated with loss of histone H3 lysine 9 (H3K9) methylation at DMR-LIT1. Histone modifications at CDKN1C promoter were not correlated with CDKN1C expression. The data suggested that the diminished CDKN1C expression is associated with the loss of methylation of CpG and H3K9 at DMR-LIT1, not by its own promoter CpG methylation, and is involved in esophageal cancer, implying that DMR-LIT1 epigenetically regulates CDKN1C expression not through histone modifications at CDKN1C promoter, but through that of DMR-LIT1.

Cell Line, Tumor↗
PubMed2006

The colorectal microRNAome.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

Base Sequence↗
PubMed2006

Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma.

A common deletion at chromosomal arm 14q32 in human renal cell carcinoma (RCC) prompted us to explore a tumor suppressor gene (TSG) in this region. We report that imprinted DLK1 at 14q32, a regulator of adipocyte differentiation, is a candidate TSG in RCCs. DLK1 expression was lost in 39 out of 50 (78%) primary RCC tissues, whereas expression of DLK1 was maintained in every normal kidney tissue examined. DLK1 was expressed in only one of 15 (7%) RCC-derived cell lines. In order to see the biological significance of DLK1 inactivation in RCCs, we tested the effect of restoration of DLK1 in RCC cell lines, using a recombinant retrovirus containing the gene. Reintroduction of DLK1 into DLK1-null RCC cell lines markedly increased anchorage-independent cell death, anoikis and suppressed tumor growth in nude mice. We then investigated the underlying mechanisms for DLK1 inactivation in RCCs. We found loss of heterozygosity at this region in 12 out of 50 RCC tissues (24%). To explore the role of epigenetic regulation of DLK1 inactivation in RCCs, we conducted methylation analysis of the upstream region and the gene body of DLK1. We could not find a differentially methylated region in either the upstream region or the gene body of DLK1. However, we found that gain of methylation upstream of GTL2, a reciprocal imprinted gene for DLK1, is a critical epigenetic alteration for the inactivation of DLK1 in RCCs. The present data have shown that gain of methylation upstream of the untranslated GTL2 leads to pathological downregulation of DLK1 in RCCs.

Animals↗
PubMed2006

Mechanisms causing imprinting defects in familial Beckwith-Wiedemann syndrome with Wilms' tumour.

The imprinted expression of the IGF2 and H19 genes is controlled by the Imprinting Centre 1 (IC1) at chromosome 11p15.5. This is a methylation-sensitive chromatin insulator that works by binding the zinc-finger protein CTCF in a parent-specific manner. Microdeletions abolishing some of the CTCF target sites (CTSs) of IC1 have been associated with the Beckwith-Wiedemann syndrome (BWS). However, the link between these mutations and the molecular and clinical phenotypes was debated. We have identified two novel families with IC1 deletions, in which individuals with the clinical features of the BWS are present in multiple generations. By analysing the methylation pattern at the IGF2-H19 locus together with the clinical phenotypes in the individuals with maternal and those with paternal transmission of five different deletions, we demonstrate that maternal transmission of 1.4-1.8 kb deletions in the IC1 region co-segregates with the hypermethylation of the residual CTSs and BWS phenotype with complete penetrance, whereas normal phenotype is observed upon paternal transmission. Although gene expression could not be assayed in all cases, the methylation detected at the IGF2 DMR2 and H19 promoter suggests that IC1 hypermethylation is consistently associated with biallelic activation of IGF2 and biallelic silencing of H19. Comparison of these deletions with a 2.2 kb one previously reported by another group indicates that the spacing of the CTSs on the deleted allele is critical for the gain of the abnormal methylation and penetrance of the clinical phenotype. Furthermore, we observe that the hypermethylation resulting from the deletions is always mosaic, suggesting that the epigenetic defect at the IGF2-H19 locus is established post-zygotically and may cause body asymmetry and heterogeneity of the clinical phenotype. Finally, the IC1 microdeletions are associated with a high incidence of Wilms' tumour, making their molecular diagnosis particularly important for genetic counselling and tumour surveillance at follow-up.

Beckwith-Wiedemann Syndrome↗
PubMed1983

Regions specifying transcriptional termination and pausing in the bacteriophage SP01 terminal repeat.

We have determined the nucleotide sequences of four termination sites recognized by Bacillus subtilis RNA polymerase. These sites are located in the terminally repeated segment of the bacteriophage SP01 genome, where most early phage transcription occurs. The SP01 terminators have structures that are similar to those recognized by Escherichia coli RNA polymerase, containing a region of dyad symmetry followed by a stretch of HMU residues in the noncoding DNA strand (HMU is substituted for T in SP01 DNA). We note that in a terminator that is only 60% efficient in vitro, there is a greater distance between these two conserved elements that exists in more efficient terminators. We also find that RNA polymerase molecules which elongate a transcript through a partial terminator often pause at this site.

Bacillus subtilis↗
PubMed2001

Human-ovine comparative sequencing of a 250-kb imprinted domain encompassing the callipyge (clpg) locus and identification of six imprinted transcripts: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8.

Two ovine BAC clones and a connecting long-range PCR product, jointly spanning approximately 250 kb and representing most of the MULGE5-OY3 marker interval known to contain the clpg locus, were completely sequenced. The resulting genomic sequence was aligned with its human ortholog and extensively annotated. Six transcripts, four of which were novel, were predicted to originate from within the analyzed region and their existence confirmed experimentally: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8. RT-PCR experiments performed on a range of tissues sampled from an 8-wk-old animal demonstrated the preferential expression of all six transcripts in skeletal muscle, which suggests that they are under control of common regulatory elements. The six transcripts were also shown to be subject to parental imprinting: DLK1, DAT, and PEG11 were shown to be paternally expressed and GTL2, antiPEG11, and MEG8 to be maternally expressed.

Animals↗
PubMed2025

LncRAnalyzer: a robust workflow for long non-coding RNA discovery using RNA-Seq.

Long non-coding RNA (lncRNA) is a major transcript category that lacks protein-coding capabilities, with relatively low abundance and complex expression patterns. Distinguishing lncRNAs from protein-coding genes is a complex process involving multiple filtering steps. We developed an automated pipeline named LncRAnalyzer featuring retrained models for 60 species. This workflow aims to reduce the likelihood of obtaining protein-coding or partial protein-coding transcripts during lncRNA identification by utilizing eight distinct approaches. We conducted a 10-fold cross-validation of the sorghum models and training sets with their standard ones and other approaches using real-life RNA-Seq datasets and known lncRNA and CDS sequences of sorghum. The results showed that the sorghum models and training sets were outperformed. The pipeline output comprises upset plots illustrating the number of lncRNA/NPCTs identified by the approaches, commonly identified lncRNA and their classes, NPCTs, and expression count tables. A feature-level comparison and benchmarking analysis of LncRAnalyzer with four existing pipelines, namely, LncPipe, LncEvo, lncRNA-Annotation, and Plant-LncPipe, demonstrated that LncRAnalyzer is more comprehensive, easier to implement, and accurate in lncRNA predictions. This workflow also ascertains lncRNA origins from various Transposable Elements (TEs) in plants using TE annotations from APTEdb [http://apte.cp.utfpr.edu.br/]. LncRAnalyzer is publicly available on GitLab [https://gitlab.com/nikhilshinde0909/LncRAnalyzer.git] for academic users.

RNA, Long Noncoding↗
PubMed2001

An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

Animals↗
PubMed1986

Activation of the cellular src gene by transducing retrovirus.

Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src protein (pp60c-src) indicated that in pp62S1-src the 8 carboxy-terminal amino acid residues of the total of 533 in pp60c-src are replaced by 43 residues translated from the env sequence at the wrong frame. In pp62S2-src, on the other hand, the 14 carboxy-terminal amino acids of pp60c-src are replaced by the 38 carboxy-terminal residues of reverse transcriptase. The mechanism of c-src transduction and the structural changes necessary for pp60c-src activation are discussed.

Animals↗
PubMed2003

CTCF binding at the insulin-like growth factor-II (IGF2)/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues.

The adjacent IGF2 and H19 genes are imprinted in most normal mouse and human tissues, but imprinting is often lost in tumors. Mouse models suggest that parental-allele specific CCCTC-binding factor (CTCF) binding at the IGF2/H19 imprinting control region (ICR) regulates the expression of these two genes. Using chromatin immunoprecipitation and PCR, we show that in several normal and neoplastic human tissues, CTCF consistently binds unmethylated ICR elements, but CTCF binding does not result in predictable gene expression. In the fetal brain, CTCF binding is monoallelic and specific for the unmethylated ICR, yet IGF2/H19 expression is biallelic. In osteosarcoma tumors, aberrant methylation of the IGF2/H19 ICR results in equally aberrant CTCF binding, yet expression of these genes does not correlate with CTCF binding. This is the first description of chromatin immunoprecipitation for CTCF binding at the human IGF2/H19 ICR, and the results demonstrate that CTCF binding at the IGF2/H19 ICR is insufficient to regulate the expression of IGF2/H19 in many human tissues.

Alleles↗
PubMed1994

Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies.

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro differentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than approximately 2000 transcripts per cell, with only modest differences between strains carrying different Xce alleles. Female embryos 7.5 days post coitum had the same number of Xist transcripts per cell as isogenic adult tissue. Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at the Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xist RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney. In addition, we found that the allelic ratio of Xist transcripts from reciprocal (XX) ES cell lines differentiating in vitro was identical to that of isogenic 10.5 to 11.5 day female embryos. These latter results suggest that there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells. However, the influence of the Xce locus on the randomness of X-inactivation in embryos seems to operate also in ES cell lines. Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating (XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.

Animals↗
PubMed1989

[Polymorphism of restriction fragments of the Ha-ras-1 protooncogene in patients with carcinoma and ulcers of the stomach].

Ha-ras restriction fragments' length polymorphism (RFLP) in white blood cells and stomach tissues from patients with carcinoma and ulcer of the stomach was examined. Genomic DNAs were digested with Xho I, Pvu II, Pst I, Msp I, Bcn I, Mva I, Bsp RI. No Ha-ras-1 polymorphic variants specifically associated with the cancer disease were detected. RFLP of the 3'-noncoding sequence of c-Ha-ras-1 gene had got features of the definite human population. The cells forming the tissue were examined and individual peculiarity of the methylated residues disposition seemed to influence RFLP of the 5'-noncoding sequence of c-Ha-ras-1.

DNA↗
PubMed1985

Characterization of the chicken aldolase B gene.

Vertebrates possess three isozyme forms of fructose diphosphate aldolase. We have isolated two overlapping chicken genomic clones which encode the liver-specific form of this enzyme; we have identified the 5' and 3' ends of this gene by a combination of primer extension analysis and S1 mapping; and we have determined the entire nucleotide sequence of this gene including 1400 base pairs (bp) of sequence from the regions flanking the 5' and 3' ends of the gene. The transcriptional unit for the aldolase B gene spans 8700 bp and contains eight intervening sequences, including a 4600-bp intron in the 5' non-coding region. On the basis of results from Southern genomic hybridizations, the aldolase B gene appears to be present only once per haploid genome. No differences were detected in the mRNA structure between RNA from three tissues expressing aldolase B (liver, kidney, and small intestine). Various features of the 5' flanking region are discussed, including a partial homology with the 5' noncoding region from rabbit aldolase A.

Animals↗
PubMed1996

IGF2 but not H19 shows loss of imprinting in human glioma.

Genomic imprinting is a gamete-specific modification resulting in the allele-specific expression of genes in somatic cells. A loss of imprinting (LOI) has been found in many embryonal and adult tumors, suggesting that it plays a role in tumor development. The incidence of LOI, however, does not seem to be ubiquitous among tumors because neuroblastoma and colorectal cancer revealed no LOI. We examined the involvement of LOI of IGF2 and H19 genes in human gliomas. The two genes were imprinted in normal brain subcortex tissues. In glioma, 8 of 14 informative cases (57%) revealed LOI in IGF2. The frequency did not depend on the tumor grade. For H19, in contrast, all 13 informative cases maintained imprinting. These results suggest that LOI of IGF2 but not H19 plays a role in the development of human glioma.

Adult↗
PubMed1998

Involvement of IGF2 and H19 imprinting in choriocarcinoma development.

Complete hydatidiform mole is an abnormal pregnancy characterized by grossly swollen villi in the absence of a fetus (Kajii and Ohama 1997, Wake et al., 1978, Jacobs et al., 1980). It is well known that this abnormal pregnancy product is androgenetic in origin. The entire genome of the molar conceptus is paternally derived. The majority of moles result from fertilization of an empty egg by haploid sperm. The paternally derived haploid set then duplicated without cytokinesis and restores diploidy. Invariably, this class of moles has a 46, XX karyotype and is completely homozygous for genetic markers. Fertilization of an empty egg by two sperms is responsible for the remaining case. These moles show a mixture of homozygous and heterozygous patterns of paternally derived genetic markers. Although complete mole is usually a benign process, 10 to 20 percent of cases leads to either invasive mole or choriocarcinoma. This propensity to malignancy has to associate with the genetic features shown in the mole, that imply the formation of homozygosity and the selective inheritance of paternal genome. It has been described in various human malignancies that both genetic features associate with the inactivation of tumor suppressor genes. Homozygosity would lead to the inactivation of tumor suppressor gene in the mole by a signal event occurred on sperm DNAs. In turn, paternal transmission would result in the silencing of particular tumor suppressor genes. Thus, tumor suppressor genes inactivated either by homozygosity formation or by paternal transmission would be involved in the pathogenesis of choriocarcinoma. Both the maternal and paternal genomes are necessary for normal embryonic development in mammals. Parental-origin-specific functional differences between two alleles, known as genomic imprinting, seem to be exploited for a regulatory mechanism crucial for the proper development of both embryonic and extraembryonic tissues. The list of imprinted genes identified is growing rapidly lately in mouse and man (DeChiara TM et al., 1991, Barlow et al., 1991, Bartolomei MS et al., 1991, Leff SE et al., 1992, Hatada I et al., 1993, Giddings SJ et al., 1994, Hayashizaki Y et al., 1994, Villar AJ et al., 1994, Guillemot F et al., 1995). Among these, IGF2 and H19 tightly linked on human chromosome 11 are of special interest because of their reciprocal imprinting and possible association with certain malignancy and congenital abnormalities. The IGF2 gene expressed from the paternally derived allele (Ohlsson et al., 1993, Giannoukalis et al., 1993), whereas the H19 gene is expressed from the maternally derived allele (Rachmilewitz et al., 1992, Zhang et al 1993, Ferguson-Smith et al., 1993).

Alleles↗
PubMed1995

The organization of the gene (EPB72) encoding the human erythrocyte band 7 integral membrane protein (protein 7.2b).

The human gene EPB72 coding for the band 7 integral membrane protein, a major protein of the erythrocyte membrane, was isolated from a genomic DNA library and characterized. Spanning approximately 30 kb, the human EPB72 gene comprises seven exons ranging from 73 to 2331 bp; intron sizes range from 970 to approximately 11,200 bp. The first exon contains the 5'-untranslated region (61 nucleotides) and the coding sequence for the N-terminal domain; the second exon encodes the hydrophobic domain, including flanking cysteine and lysine residues. Exon 7 contains the C-terminal portion and a 2-kb 3'-untranslated region. The potential promoter region contains several consensus sequences for ubiquitous transcription factors (Sp1, AP1, AP2, CP1/2, NF kappa B, CREB, Ets-1, and CACC/GT-BF) and two imperfect sequences for erythroid factors (EKLF and GATA-1), in accordance with the ubiquitous distribution of the EPB72 mRNA in different cell types. No TATA box was apparent. An inverted Alu repeat element, flanked by nonamer direct repeats, was identified within the region -913/-620, relative to the cap site. Six additional Alu repeat elements, including one monomer and one trimer, were identified within the introns and the 3'-untranslated region. Two polyadenylation signals in the 3'-noncoding region of exon 7 enable the production of two mRNA species.

Amino Acid Sequence↗
PubMed2000

Microsatellites in different eukaryotic genomes: survey and analysis.

We examined the abundance of microsatellites with repeated unit lengths of 1-6 base pairs in several eukaryotic taxonomic groups: primates, rodents, other mammals, nonmammalian vertebrates, arthropods, Caenorhabditis elegans, plants, yeast, and other fungi. Distribution of simple sequence repeats was compared between exons, introns, and intergenic regions. Tri- and hexanucleotide repeats prevail in protein-coding exons of all taxa, whereas the dependence of repeat abundance on the length of the repeated unit shows a very different pattern as well as taxon-specific variation in intergenic regions and introns. Although it is known that coding and noncoding regions differ significantly in their microsatellite distribution, in addition we could demonstrate characteristic differences between intergenic regions and introns. We observed striking relative abundance of (CCG)(n)*(CGG)(n) trinucleotide repeats in intergenic regions of all vertebrates, in contrast to the almost complete lack of this motif from introns. Taxon-specific variation could also be detected in the frequency distributions of simple sequence motifs. Our results suggest that strand-slippage theories alone are insufficient to explain microsatellite distribution in the genome as a whole. Other possible factors contributing to the observed divergence are discussed.

Animals↗

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