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PubMed1998

Molecular correlates of the murine Xce locus.

The murine Xce locus, first identified by Bruce Cattanach, influences the primary choice of the X chromosome to be inactivated. Methylation of a GC-rich region (DXPas34) that includes multiple 34 bp repeats and lies some 15 kb 3' to Xist has been shown to vary with Xce haplotype. The degree of methylation on the active X chromosome at this locus represents one of the few molecular correlates of Xce action currently available. Data relating to the specificity and other characteristics of this association are presented.

Animals↗
PubMed2000

Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Animals↗
PubMed2005

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases.

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.

Animals↗
PubMed1998

Patterns of ENOD40 gene expression in stem-borne nodules of Sesbania rostrata.

At the base of adventitious root primordia, located on the stem of the tropical legume Sesbania rostrata, nitrogen-fixing nodules are formed upon inoculation with the microsymbiont Azorhizobium caulinodans. This pattern of nodule development presents features of indeterminate and determinate nodules in early and later stages, respectively. A S. rostrata cDNA clone homologous to early nodulin ENOD40 genes was isolated from a cDNA library of developing stem nodules. SrENOD40-1 contained the conserved regions I and II of other ENOD40 genes. By reverse transcriptase PCR, enhanced SrENOD40-1 expression was observed in the adventitious root primordia between 4 and 8 h after inoculation with A. caulinodans. In situ hybridization showed that SrENOD40-1 transcripts, present around the central vascular bundle of the uninfected root primordia, were strongly enhanced upon induction of nodule development. De novo SrENOD40-1 expression was observed in the initiating and growing nodule primordia and around vascular bundles. When cell type specification sets in, the expression became pronounced in cells derived from the meristematic regions. In other parts of the plant, weak SrENOD40-1 expression was associated with vascular bundles and was observed in leaf and stipule primordia.

Amino Acid Sequence↗
PubMed1999

The soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially regulated.

The ENOD40 gene is induced early during Rhizobium-legume symbiosis and has probably a primary role in the nodule organogenesis. In this paper we show that the 1.7 kb 5'-flanking region of the GmENOD40(2) is able to drive the expression of a gusA-int marker in transgenic Arabidopsis thaliana. The promoter activity is developmentally regulated and the major activity is detected in the root and in the stigma.

Arabidopsis↗
PubMed2000

Characterization of human and mouse H19 regulatory sequences.

H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.

Animals↗
PubMed1998

Comparative mapping of X chromosomes in vole species of the genus Microtus.

Comparative mapping of X-linked genes has progressed rapidly since Ohno's prediction that genes on the X chromosome should be conserved as a syntenic group in all mammals. Although several conserved blocks of homology between human and mouse have been discovered, rearrangements within the X chromosome have also been characterized. More recently, some exceptions to Ohno's law have been reported. We have used fluorescence in situ hybridization (FISH) to map five genes, Gla, G6pd, Hprt, Pgk1 and Xist, to two of the largest conserved segments of X material in five members of the genus Microtus (grey vole) and show that vole X chromosomes demonstrate greater homology to human than to mouse. Cytogenetic analysis indicates a relatively high frequency of rearrangement during vole evolution, although certain blocks of homology appear to be highly conserved in all species studied to date. On this basis we were able to predict the probable location of the rat X inactivation centre (Xic) based solely on high-resolution G-banding. Our prediction was then confirmed by mapping the rat Xist gene by FISH. The possible significance of conserving long-range chromosome structure in the vicinity of the Xic is discussed with respect to the mechanism of X inactivation.

Animals↗
PubMed2002

Genetic and clinical analysis of spinocerebellar ataxia type 8 repeat expansion in Yugoslavia.

Spinocerebellar ataxia type 8 (SCA8) is a slowly progressive ataxia causally associated with untranslated CTG repeat expansion on chromosome 13q21. However, the role of the CTG repeat in SCA8 pathology is not yet well understood. Therefore, we studied the length of the SCA8 CTA/CTG expansions (combined repeats, CRs) in 115 patients with ataxia, 64 unrelated individuals with non-triplet neuromuscular diseases, 70 unrelated patients with schizophrenia, and 125 healthy controls. Only one patient with apparently sporadic ataxia was identified with an expansion of 100 CRs. He had inherited the expansion from his asymptomatic father (140 CRs) and transmitted the mutation to his son (92 CRs). Paternal transmission in this family produced contractions of 40 and 8 CRs, respectively. None of the subjects from other studied groups had an expansion at the SCA8 locus. In the control group the number of CRs at the SCA8 locus ranged from 14 to 34. Our findings support the notion that allelic variants of the expansion mutation at the SCA8 locus can predispose to ataxia.

Genes, Dominant↗
PubMed2002

SCA8 in the Spanish population including one homozygous patient.

Controversial data have been reported about SCA8 since its description in 1999. The most accepted hypothesis is that CTG expansions within the CTA/CTG combined repeat expansion in the SCA8 locus causes SCA8. It is inherited as a dominant trait with reduced penetrance. The present study, reports the first data regarding SCA8 in the Spanish population and the clinical findings in patients carrying expanded alleles, including one homozygous patient. Two hundred and forty-six individuals from the Spanish population, including controls (149) and ataxic patients (97), were studied. DNA was extracted from blood samples using standard methods. Amplification of the CTA/CTG 3'untranslated region was achieved by PCR using primers SCA8-F3 and SCA8-R4 and conditions described previously. Neurological reevaluation was done in individuals carrying the expanded allele. We detected five unrelated expanded alleles corresponding to three affected patients (one of them homozygous) and one healthy individual. SCA8 represents 4% of the total dominant spinocerebellar ataxias studied in our group (Spanish population) (three index patients out of 75 dominant ataxic independent nucleus). The patient that resulted homozygous for the expansion is a 25-year-old man with a clinical picture of progressive ataxia and dysarthria that began at the age of 12. On neurological examination, he showed ataxia, slight dysarthria and nystagmus to the extreme lateral gaze. A cranial MRI showed global atrophy of cerebellum but the brainstem was spared. Family history showed the presence of ataxia in his grandfather and father. His mother is healthy at the age of 52 and a molecular study of SCA8 reveals one allele that could be considered as premutated. She has no ataxia antecedents in her family. Our results provide additional information about the SCA8 expansion, within the Spanish population. These results are in agreement with the hypothesis of the CTG expansion in the SCA8 locus being responsible for the SCA8 ataxia showing reduced penetrance. Besides homozygous status, advancing age at onset (as previously described for other SCAs) supports this idea.

Adult↗
PubMed2000

Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene.

The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.

Animals↗
PubMed2000

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

Animals↗
PubMed2001

X-chromosome inactivation: counting, choice and initiation.

In many sexually dimorphic species, a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. In mammals, such dosage compensation is achieved by X-chromosome inactivation, a process that presents a unique medley of biological puzzles: how to silence one but not the other X chromosome in the same nucleus; how to count the number of X's and keep only one active; how to choose which X chromosome is inactivated; and how to establish this silent state rapidly and efficiently during early development. The key to most of these puzzles lies in a unique locus, the X-inactivation centre and a remarkable RNA--Xist--that it encodes.

Animals↗
PubMed1998

Location of enhancers is essential for the imprinting of H19 and Igf2 genes.

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.

Alleles↗
PubMed1994

The human X-inactivation centre is not required for maintenance of X-chromosome inactivation.

X-chromosome inactivation occurs early in mammalian female development to achieve dosage compensation with males. Although it is widely accepted that this inactivation requires the presence in cis of the X-inactivation centre (XIC), it is not known whether the XIC is required for the initiation, promulgation or maintenance of X inactivation. The XIST gene, which is localized within the XIC interval on both the human and mouse X chromosomes, is constitutively expressed from inactive X chromosomes, suggesting a possible role in the maintenance of X inactivation. To address whether the presence of the XIC, including the XIST gene, is continuously required for the maintenance of X-chromosome inactivation, we have analysed the transcriptional activity of a number of X-linked genes in mouse/human somatic cell hybrids retaining an intact human inactive X chromosome or derivatives of the inactive X chromosome lacking the XIC. Genes subject to X inactivation remain transcriptionally silent despite the loss of the XIC, demonstrating that the presence of the XIC is not required for the maintenance of X inactivation in somatic cells.

Animals↗
PubMed1996

Requirement for Xist in X chromosome inactivation.

The Xist gene has been proposed as a candidate for the X inactivation centre, the master regulatory switch locus that controls X chromosome inactivation. So far this hypothesis has been supported solely by indirect evidence. Here we describe gene targeting of Xist, and provide evidence for its absolute requirement in the process of X chromosome inactivation.

Alleles↗