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PubMed1997

A methylation-dependent DNA-binding activity recognising the methylated promoter region of the mouse Xist gene.

Differential methylation of CpG sites in the promoter region of the mouse Xist gene is correlated with Xist expression and X-chromosome inactivation in the female. Using oligonucleotides encompassing the differentially methylated sites as probes in band-shift assays, we have identified a nuclear protein which binds to a specific region of the promoter (between base pairs -45 and -30 upstream from the transcription start site) only when CpG sites within the CG rich region (GCGCCGCGG, -44 to -36) are methylated. Competition experiments with methylated or unmethylated heterologous oligonucleotides demonstrate that the activity is sequence-specific as well as methylation-dependent. Analysis by Southwestern blot identifies a protein of approximately 100 kDa molecular weight and confirms strong binding to the methylated Xist promoter oligonucleotide. Using a 233bp Xist-promoter luciferase construct in which the cytosines in the three CpG sites in the -44 to -36 region are mutated to thymine, we have established that this region is required for transcription from the mouse Xist promoter. Therefore, we suggest that the binding of the 100kDa protein to the methylated sequence leads to repression of transcription from the methylated Xist allele, thus suggesting a role in the regulation of both imprinted and random Xist transcription and X-chromosome inactivation.

Animals↗
PubMed1997

Mutational analysis reveals that an array of GCAAG/CTTGC motifs between sprit promoter sequences for RNA polymerase III is essential for neural BC1 RNA transcription.

BC1 RNA is expressed from an identifier (ID) sequence by RNA polymerase III (Pol III) and occurs in neural cells as a ribonucleoprotein particle (BC1 RNP). On the BC1 RNA gene, between the Pol III promoter A and B boxes, there is a region which contains short inverted repeats, including three GCAAG/CTTGC motifs. We found that a nuclear protein binds specifically to this region and, using an in vitro transcription system, demonstrated that point mutations within these motifs markedly inhibit BC1 RNA transcription. These results suggest that the GCAAG/CTTGC motif region and its binding protein may play a role in the transcription of BC1 RNA. Moreover, we demonstrated that transcription is repressed by a concomitant molar excess of BC1 RNA and that the BC1 RNA transcribed by this system forms an RNP with nuclear protein(s), suggesting some interaction of BC1 RNA with transcription factor(s).

Animals↗
PubMed1998

Difference in chromatin packaging between active and inactive X chromosomes by fractionation and allele-specific detection.

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

Alleles↗
PubMed1998

An E-box sequence acts as a transcriptional activator for BC1 RNA expression by RNA polymerase III in the brain.

BC1 RNA is a small cytoplasmic RNA that is transcribed by RNA polymerase III (Pol III) in the rodent nervous system. In addition to essential intragenic promoter elements for Pol III, the BC1 RNA gene has five E-box sequences (CANNTG) in its 5' flanking region. Deletion analysis using an in vitro transcription system revealed that the region containing the E2 site (CAATTG) was necessary for effective transcription of BC1 RNA. A construct with point mutations within the E2 site showed reduced transcriptional activity. Furthermore, DNaseT I protection and gel retardation assays demonstrated that the E2 site was recognized specifically by a brain nuclear protein(s). These results suggest that the upstream E-box sequence and its binding protein may be involved in the regulation by Pol III of preferential BC1 RNA expression in the brain.

Animals↗
PubMed1998

BC1 RNA protein particles in mouse brain contain two y-,h-element-binding proteins, translin and a 37 kDa protein.

Neural BC1 RNA is distributed in dendrites in the form of ribonucleoprotein particles (RNP). Recently, Han et al. reported that testis-brain RNA-binding protein (the mouse homologue of Translin), known to be a translational repressor, links a subset of mRNAs to microtubules (MTs) through binding to their y-,h-sequence elements. We found that similar elements are also present in BC1 RNA. Therefore, it is possible that they also act as cis-elements, together with their transacting proteins, and control the transport of BC1 RNA along dendritic MTs. In this study, we demonstrated that two y-,h-element-binding proteins copurified with BC1 RNP. One was identified as mouse Translin and the other was a 37 kDa protein. This macromolecular assembly may constitute a transport particle for BC1 RNA. It is also possible that BC1 RNP or Translin itself may play a regulatory role(s) in the translation of mRNAs within dendrites.

Animals↗
PubMed1998

The dendritic translocation of translin protein in the form of BC1 RNA protein particles in developing rat hippocampal neurons in primary culture.

Neural BC1 RNA is distributed in neuronal dendrites as ribonucleoprotein particles (RNP). Our previous studies indicated the presence of Translin in BC1 RNPs, which is a translational repressor and links a subset of mRNAs to microtubules. In this study, we confirmed that Translin associates with BC1 RNP and we used immunocytochemical methods to examine the subcellular distribution of Translin in developing hippocampal cells in primary cultures. Translin was detected in both the nuclei and cytoplasm of neurons, whereas in glial cells it was localized in the nuclei. Consistent with the reported developmental time course of BC1 RNA expression and dendritic delivery the translocation of Translin to the neuronal dendrites appeared to correlate with neuronal development and differentiation events such as the onset of synaptogenesis in culture. These observations suggest that BC1 RNP or Translin itself may be relevant to the dendritic translation of mRNAs in response to transsynaptic activity.

Animals↗
PubMed1999

Proinsulin-like growth factor-II overexpression does not alter monoallelic H19 gene expression in transfected human embryonic kidney fibroblasts.

Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. The H19 gene is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in tumorigenesis. The H19 gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues. H19 is expressed only from the maternal and IGF2 from the paternal chromosome. We have asked whether overexpression of proIGF-II alters H19 imprinting status and/or expression. Human embryonal kidney fibroblasts (293 cells) were stably transfected with a PCMV5 vector containing the full length human IGF-II cDNA or a control cDNA. Transfectant clones expressed large quantities of IGF-II mRNA and secrete 1-5 ug/ml and 150-230 ng/ml proIGF-II within 24 hours of serum-free culture (transfectant 293-9 and -11 respectively) (1). Cells were genotyped at the exon 5, RsaI restriction fragment length polymorphism (RFLP) and found to be informative (+/-). H19 expression was monoallelic (+) indicating preservation of H19 imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression of IGF2 in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect to H19 expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express the H19 gene. (2) H19 imprinting is preserved in these cells. (3) proIGF-II overexpression does not alter H19 expression.

Alleles↗
PubMed1999

An antisense transcript to SMAD5 expressed in fetal and tumor tissues.

SMAD5, a transducer of TGF-beta/BMP inhibitory signals and a tumor suppressor candidate, localizes to the region of invariant loss in human myeloid neoplasms, on chromosome 5q31.1. Recent evidence indicates a gene-dosage effect along the TGF-beta/BMP signaling pathways. We have identified a novel transcript designated DAMS, whose 3' exonic sequences contain in part an alternate 5' exon of SMAD5, in the antisense orientation. Expressed sequenced tags (ESTs) for DAMS are found in fetal tissues (heart, adrenal glands, and total fetus) and pancreatic tumor cDNA libraries. In contrast to SMAD5, DAMS expression is not readily detectable in adult and fetal tissues. Semiquantitative PCR suggests that the stoichiometry between SMAD5 and DAMS transcripts ranges between 15 and 120 in normal and malignant hematopoietic cells. The findings raise the possibility that DAMS may be a fail-safe mechanism for precise regulation of SMAD5 transcript levels that may be critical in maintaining normal homeostasis.

5' Untranslated Regions↗
PubMed1999

Molecular cloning of antisense transcripts of the mouse Xist gene.

Prior to X-inactivation, Xist is transcribed in unstable form. The initiation of X-inactivation is associated with the appearance of stable Xist transcripts which coat the X chromosome to be inactivated. Using strand specific RT-PCR analysis of the 5' region of Xist, we have detected antisense transcripts (Xist AS) in undifferentiated embryonic stem (ES) cells, but not in female somatic cells. Screening of a female ES cell cDNA library allowed us to isolate one poly(A)-tailed cDNA clone corresponding to this RNA. 5' RACE analysis showed that XistAS and the P1 sense product of Xist overlap by at least 707 bp. Expression of XistAS was also detected in early mouse embryos before random X-inactivation in the epiblast lineage. Although XistAS is low in abundance, it may be involved in destabilizing Xist mRNA in undifferentiated ES cells.

Animals↗
PubMed2000

Neural BC1 RNA associates with pur alpha, a single-stranded DNA and RNA binding protein, which is involved in the transcription of the BC1 RNA gene.

BC1 RNA is preferentially expressed in neural cells by RNA polymerase III (Pol III) and forms ribonucleoprotein particles (RNP) in the somatodendritic domain of neurons. Our previous studies have suggested that, in the nucleus, BC1 RNA forms an RNP containing a nuclear protein(s) that participates in the transcription of the BC1 RNA gene. In this study, we have shown that newly synthesized BC1 RNA in purified brain nuclear extracts is immunoprecipitated by an antibody against Pur alpha. Pur alpha is a protein that binds single-stranded DNA and RNA and is known to regulate transcription of Pol II system. Although BC1 RNA is transcribed by Pol III, the BC1 RNA gene has two putative Pur alpha binding sites, which Pur alpha specifically recognizes. Point mutations within these sites reduced transcriptional activity in vitro. Furthermore, transcription was inhibited by depletion of Pur alpha from the nuclear extracts, either by the coexistence of its binding region of BC1 RNA or by the antibody that was able to precipitate the nuclear BC1 RNP. These observations suggest that BC1 RNA associates with Pur alpha which is involved in the transcription of the BC1 RNA gene.

Animals↗
PubMed1995

Developmental regulation of genomic imprinting during gametogenesis.

Successful mammalian development requires both the male and female genomes. This is due in part to genomic imprinting, which results in offspring inheriting only one functional copy of a gene from either the mother or the father. Evidence suggests that this specialization of the parental genomes is established during gametogenesis when the imprint pattern inherited from the parent is switched to reflect the sex of the progeny. We used reverse transcription-PCR to analyze the allele-specific expression of Igf-2, Igf-2r, and H19 in the testes and ovaries of mice derived from an interspecies cross between Mus musculus and Mus spretus. Because of genomic imprinting, Igf-2 is expressed only from the paternal allele and Igf-2r and H19 only from the maternal allele, in most tissues. Although allele-specific expression was maintained in the neonatal testis and ovary, relaxation of imprinting was detected by 7 days after birth in the male and continued during testis development. In the female, relaxation of the Igf-2 and Igf-2r parental imprints was observed in the adult ovary and oocyte. These results (1) indicate that imprinted expression is relaxed during gametogenesis, presumably as a consequence or prerequisite of the imprinting mechanism, and (2) predict a subsequent imprinting event after which the allele-specific expression of Igf-2, Igf-2r, and H19 reflects the parent of origin.

Alleles↗
PubMed1996

X-Inactivation and histone H4 acetylation in embryonic stem cells.

In female mammalian cells, dosage compensation for X-linked genes is achieved by the transcriptional silencing, early in development, of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression of Xist, a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. We describe here the use of these cells in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, we show that an underacetylated X chromosome appears only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by Day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by Day 4 of differentiation (i.e. , when deacetylation of Xi is first seen). The overall deacetylation of Xi follows Xist expression and the first appearance of a single, late-replicating X, both of which occur on Day 2. It also follows the silencing of X-linked genes. Levels of mRNA from four such genes, Hprt, G6pd, Rps4, and Pgk-1, had all fallen by approximately 50% (relative to the autosomal gene Aprt) by Days 2-4. The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, we note that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach we have used and that such selective deacetylation may still be part of the silencing process.

Acetylation↗
PubMed2002

Mouse parthenogenetic embryos with monoallelic H19 expression can develop to day 17.5 of gestation.

In mammals, both maternal and paternal genomes are required for a fetus to develop normally to term. This requirement is due to the epigenetic modification of genomes during gametogenesis, which leads to an unequivalent expression of imprinted genes between parental alleles. Parthenogenetic mouse embryos that contain genomes from nongrowing (ng) and fully grown (fg) oocytes can develop into 13.5-day-old fetuses, in which paternally and maternally expressed imprinted genes are expressed and repressed, respectively, from the ng oocyte allele. The H19 gene, however, is biallelically expressed with the silent status Igf2 in such parthenotes. In this study, we examined whether the regulation of H19 monoallelic expression enhances the survival of parthenogenetic embryos. The results clearly show that the ng(H19-KO)/fg(wt) parthenogenetic embryos carrying the ng-oocyte genome that had been deleted by the H19 transcription unit successfully developed as live fetuses for 17.5 gestation days. Control experiments revealed that this unique phenomenon occurs irrespective of the genetic background effect. Quantitative gene expression analysis showed that day 12.5 ng(H19-KO)/fg(wt) parthenogenetic fetuses expressed Igf2 and H19 genes at <2 and 82% of the levels in the controls. Histological analysis demonstrated that the placenta of ng(H19-KO)/fg(wt) parthenotes was afflicted with atrophia with severe necrosis and other anomalies. The present results suggest that the cessation of H19 gene expression from the ng-allele causes extended development of the fetus and that functional defects in the placenta could be fatal for the ontogeny.

Alleles↗
PubMed1996

Activation of the inactive X chromosome induced by cell fusion between a murine EC and female somatic cell accompanies reproducible changes in the methylation pattern of the Xist gene.

Mouse embryonal carcinoma (EC) cell lines are divided into two classes with or without the capability of reactivating the inactive X chromosome from a fusion partner of female lymphocyte. The 5' region of Xist was partially methylated in reactivating-competent EC cells but was fully methylated in reactivating-incompetent EC cells having a single X chromosome. Partial or heterogeneous methylation implies methylation of each CpG site in about half of the cell independently of methylation status of neighboring CpG sites. Fusion of the reactivating-competent EC cells with female lymphocytes induced not only de novo methylation in the 5' region of Xist allele on the hitherto inactivated X chromosome, but also demethylation of the same region of Xist on the other X chromosome from the female somatic cell. In contrast, no such changes occurred in hybrid cells involving reactivating-incompetent EC cells. Thus, partial methylation of the 5' region of Xist most probably maintained by low maintenance and high de novo methylation efficiency is correlated with reactivation potential of the EC cell. It is possible that this unique methylation pattern is implicated in random X inactivation in EC-hybrid cells in vitro and in epiblast cells in vivo.

Animals↗
PubMed1997

Reactivation of an inactive human X chromosome introduced into mouse embryonal carcinoma cells by microcell fusion with persistent expression of XIST.

An inactive human X chromosome was introduced by microcell fusion into two mouse embryonal carcinoma cell lines, PSA1-TG8 and OTF9-63, each of which has a single X chromosome. The donor cell line was a mouse-human somatic cell hybrid, CF150, retaining one or more inactive human X chromosome(s) per cell as its only human element. Twenty hybrid clones isolated retained EC morphology and contained the intact human X chromosome(s) or its truncated derivative(s). Replication banding analysis showed that the introduced human X chromosome(s) or its derivative(s) replicated synchronously with other mouse chromosomes, suggesting reactivation of the human X chromosomal elements after transfer. Reversal of inactivation was further confirmed by the expression of five human X-linked genes repressed in CF150, although the XIST (X inactive specific transcript) gene continued to be active. The level of XIST expression in our hybrid cells was almost identical to that of parental CF150 cells. Methylation status of 5' end of the active XIST gene varied considerably from almost full methylation to unmethylation in these hybrids. Thus, mouse EC cells used in this study were capable of altering methylation status of the human XIST gene in a manner lacking consistency and unable to repress its transcription. Furthermore, we failed to obtain any positive evidence for the occurrence of X chromosome inactivation in differentiating monochromosome EC hybrids. Taken together, these findings suggest that the human X chromosome inactivation center including the XIST gene is unable to function effectively in mouse cells.

Animals↗
PubMed1997

Different strategies of X-inactivation in germinal and somatic cells: histone H4 underacetylation does not mark the inactive X chromosome in the mouse male germline.

It has previously been shown by immunocytochemistry that the inactive X chromosome (Xi) in somatic cells of human and mouse females is marked by underacetylation of histone H4. It has been suggested that this may be important for transcriptional silencing of genes on Xi. We have now investigated X-inactivation in meiotic cells of the male germline. In these cells the single X chromosome is transcriptionally inactive and expresses XIST, a gene that in somatic cells is transcribed only from Xi. By immunostaining with antibodies to H4 acetylated at lysines 5, 8, 12, or 16, we demonstrate that histone H4 on the male X is not underacetylated. We conclude that there is a differential germline strategy for maintenance of X-inactivation and that H4 underacetylation, though associated with the long-term marking of inactive X chromosomes in the female soma, is not always essential for the transcriptional down-regulation of X-linked genes.

Acetylation↗
PubMed1999

Monoallelic expression of reactivated imprinted genes in embryonal carcinoma cell hybrids.

Though DNA methylation is necessary to maintain monoallelic expression of imprinted genes, it is still unclear whether it represents the primary mark. Here we ask whether the imprinting mark is still present in terminally differentiated somatic cells in which the transcription of embryo-specific imprinted genes was shut off. For such analysis H19 and Igf2 genes were activated by inducing differentiation of (mouse embryonal carcinoma cell x mouse lymphocyte) hybrid cell clones. Although lymphocytes do not express H19 and Igf2, both genes are reactivated in a proper monoallelic manner in hybrid cells. Analysis of the upstream region of the H19 gene confirmed maintenance of differential methylation of the active and inactive H19 genes of lymphocyte origin, although a tendency toward in vitro induced hypermethylation was apparent. We conclude that the imprints of the H19, U2af1-rs1, and Igf2 genes are maintained in lymphocytes in adult mice.

Alleles↗
PubMed2002

Cross-talk between mesenchyme and epithelium increases H19 gene expression during scattering and morphogenesis of epithelial cells.

The H19 gene is an imprinted gene expressed from the maternal allele. It is known to function as an RNA molecule. We previously reported that in breast adenocarcinoma, H19 is often overexpressed in stromal cells and preferentially located at the epithelium/stroma boundary, suggesting that epithelial/mesenchymal interactions can control H19 RNA expression. In some cases of breast adenocarcinoma with poor prognosis, H19 is overexpressed in epithelial cells. Therefore we examined whether mesenchymal factors can induce H19 expression in epithelial cells. Using quantitative RT-PCR and in situ hybridization, we found that when mammary epithelial cells were cultured in collagen gels, H19 expression was strongly up-regulated compared to when cells were cultured on plastic. Collagen gels allow three-dimensional growth of epithelial cells and morphogenetic responses to soluble factors. A conditioned medium from MRC-5 fibroblasts caused branching morphogenesis of HBL-100 cells and invasive growth of MDA-MB-231 cells, whereas MCF-7 cells were unresponsive. Induction of H19 expression correlated with morphological changes in HBL-100 and in MDA-MB-231 cells, whereas H19 expression was not induced in MCF-7 cells. Using a blocking antibody, HGF/SF was identified as the fibroblast-derived growth factor capable of inducing H19 expression and cell morphogenesis. We further demonstrated that H19 promoter activity was stimulated by various growth factors using transient transfection in MDCK epithelial cells. HGF/SF was more efficient than EGF or FGF-2 in transactivating the H19 promoter, whereas IGF-2, TGFbeta-1, and TNF-alpha were ineffective. This activation by HGF/SF was prevented by pharmacological inhibition of MAP kinase or of phospholipase C. We conclude that H19 is a target gene for HGF/SF, a known regulator of epithelial/mesenchymal interactions, and suggest that the up-regulation of H19 may be implicated in morphogenesis and/or migration of epithelial cells.

Animals↗