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Results for “Locus Control Region”

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PubMed2005

Developmental regulation of the beta-globin gene locus.

The beta-globin genes have become a classical model for studying regulation of gene expression. Wide-ranging studies have revealed multiple levels of epigenetic regulation that coordinately ensure a highly specialised, tissue- and stage-specific gene transcription pattern. Key players include cis-acting elements involved in establishing and maintaining specific chromatin conformations and histone modification patterns, elements engaged in the transcription process through long-range regulatory interactions, transacting general and tissue-specific factors. On a larger scale, molecular events occurring at the locus level take place in the context of a highly dynamic nucleus as part of the cellular epigenetic programme.

Animals↗
PubMed2004

Overexpression of cyclooxygenase-2 correlates with chromosomal gain at the cyclooxygenase-2 locus and decreased patient survival in advanced colorectal carcinomas.

PURPOSE: Overexpression of cyclooxygenase-2 is observed in a variety of malignancies including colorectal cancer. However, to date, cyclooxygenase-2 expression by advanced human colorectal cancers and their metastases has been poorly characterized. This study was designed to evaluate the rate of cyclooxygenase-2 overexpression in our tumor collection and to clarify its correlation with the chromosomal status at the cyclooxygenase-2 locus in colorectal cancer. METHODS: Seventy-four specimens were analyzed immunohistochemically using a monoclonal cyclooxygenase-2 antibody. The staining was scored semiquantitatively as: -, negative; +, weak; ++, moderate; and +++, strong positive. Of these, 45 specimens were analyzed using comparative genomic hybridization and immunohistochemistry. We correlated the cyclooxygenase-2 overexpression with the chromosomal gain of 1q25.2-q25.3 and patients survival and compared primary colorectal cancers and their paired metastases at the DNA and protein level. RESULTS: Overexpression was observed in 58 percent of the cases (score > or = ++). Chromosomal gains at the cyclooxygenase-2 locus were clearly correlated with overexpression of the gene (P=0.009). Furthermore, the comparison of paired tumor samples showed additional overrepresentation in the metastases at the cyclooxygenase-2 locus, which could be confirmed by immunohistochemistry. Kaplan-Meier analysis showed that overexpression of cyclooxygenase-2 was significantly associated with poor survival and thus could serve as a prognostic marker. CONCLUSIONS: We conclude that cyclooxygenase-2 is related with tumor progression and metastasis in colorectal cancer, which can be observed on protein level, and reflects chromosomal gain at the locus at 1q25.2-q25.3.

Chromosome Aberrations↗
PubMed2006

Genome-wide scanning of HoxB1-associated loci in mouse ES cells using an open-ended Chromosome Conformation Capture methodology.

Spatial proximity between genomic loci can play important roles in their function and regulation. We have developed an open-ended method based on Chromosome Conformation Capture technology allowing us to perform genome-wide scanning of the loci that form the spatial environment of a given locus at a given time. As a proof of principle we present the use of this methodology to investigate the dynamics of the spatial environment of the HoxB1 gene before and after the induction of its expression in mouse embryonic stem cells. Our results indicate that the HoxB1 locus' immediate spatial environment can be divided roughly into three parts: a first part is represented by a domain of immediate proximity on each side of the HoxB1 locus covering approximately 110 kb, a second part extends to a domain of 800 kb and a third part consists of distal intra-chromosomal and inter-chromosomal interactions. Consistent with FISH studies showing the decondensation and repositioning of HoxB1 outside of its chromosomal territory during its expression, the proportion of inter-chromosomal interactions between HoxB1 and the rest of the genome increases after its induction, while interactions with distal intra-chromosomal loci become less frequent. These results indicate that this technique can be used to determine the dynamics of loci interactions on a genome-wide scale.

Animals↗
PubMedDate not supplied

Transcriptional potentials of the beta-like globin genes at different developmental stages in transgenic mice and hemoglobin switching.

Developmental-stage-specific regulation and physiological levels of expression of the globin genes can be recaptured in transgenic mice carrying a YAC/BAC- or cosmid-based construct. By contrast, proper developmental regulation and high-level expression cannot be achieved coordinately in transgenic mice carrying a more manipulated construct, such as a plasmid-based globin gene construct. These differences provide us an opportunity to define the requirements for a developmentally regulated, high-level expression of the globin genes in vivo. To achieve this, as a first step, we studied maximum transcriptional potentials of the beta-globin genes at various stages of development. microLCR-enhanced expression of the epsilon-, gamma-, and beta-globin genes driven by their minimal promoters was estimated and compared with that in betaYAC transgenic mice. Quantitative measurements of steady state mRNA levels of the epsilon-, gamma-, and beta-globin genes showed that the microLCR was able to enhance expression of each beta-like globin gene to levels similar to those in the betaYAC mice. Moreover, transcriptional potentials of each globin gene were unchanged during the entire course of development. These observations indicate that the highest level of expression of the globin genes can be achieved in both embryonic and definitive erythropoiesis regardless of developmental specificity of the genes. This finding implies that transcription suppression is the major mechanism of the developmental specificity of the expression of the beta-like globin genes.

Animals↗
PubMed2005

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.

Animals↗
PubMed2005

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library.

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.

Animals↗
PubMed2006

Chromatin looping and the probability of transcription.

Recent studies of several multigene clusters have shown that gene activation by a remote enhancer is associated with chromatin loop formation. It is not fully understood how a chromatin loop forms in a nucleus or how it is involved in gene regulation. In this article, we propose that the major feature that determines loop formation is the flexibility of chromatin, and that this flexibility is modulated by histone acetylation (and other modifications). Thus, histone modifications will modulate distribution of the preferential looping site in chromatin, which, in turn, determines the probability of interaction between a remote enhancer and the cognate genes. This model can explain gene expression changes in the Hoxd gene cluster and the beta-globin locus.

Acetylation↗
PubMed2002

Chromatin boundaries in budding yeast: the nuclear pore connection.

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Active Transport, Cell Nucleus↗
PubMed2001

Independent patterns of transcription for the products of the rat cholinergic gene locus.

The cholinergic phenotype requires the expression of the vesicular acetylcholine transporter and choline acetyltransferase proteins. Both genes are encoded at one chromosomal location called the cholinergic gene locus. We have identified by in situ hybridization histochemistry distinct patterns of transcription from the cholinergic gene locus in the subdivisions of the rat cholinergic nervous system. The vesicular acetylcholine transporter and choline acetyltransferase are co-expressed in cholinergic neurons at all developmental stages in all major types of cholinergic neurons. The relative levels of vesicular acetylcholine transporter and choline acetyltransferase transcripts, however, change substantially during development in the CNS. They also differ dramatically in distinct subdivisions of the mature cholinergic nervous system, with vesicular acetylcholine transporter mRNA expressed at high levels relative to choline acetyltransferase mRNA in the peripheral nervous system, but at equivalent levels in the CNS. Expression of the R-exon, the presumptive first non-coding exon common to both the vesicular acetylcholine transporter and choline acetyltransferase, was not detectable at any developmental stage in any of the cholinergic neuronal subtypes in the rat nervous system. Thus, in contrast to less complex metazoan organisms, production of the vesicular acetylcholine transporter and choline acetyltransferase via a common differentially spliced transcript does not seem to occur to a significant extent in the rat. We suggest that separate transcriptional start sites within the cholinergic gene locus control vesicular acetylcholine transporter and choline acetyltransferase transcription, while additional elements are responsible for the specific transcriptional control of the entire locus in cholinergic versus non-cholinergic neurons. Independent transcription of the vesicular acetylcholine transporter and choline acetyltransferase genes provides a mechanism for regulating the relative expression of these two proteins to fine-tune acetylcholine quantal size in different types of cholinergic neurons, both centrally and peripherally.

Acetylcholine↗
PubMed2003

Primary pulmonary hypertension may be a heterogeneous disease with a second locus on chromosome 2q31.

OBJECTIVES: The aim of our study was to identify genetic causes of primary pulmonary hypertension (PPH), to estimate the proportion of families with mutations in the BMPR2 (bone morphogenetic protein receptor type 2) gene, and to examine whether genetic heterogeneity might play a role. BACKGROUND: The BMPR2 mutations have been identified in a substantial portion of patients with familial or sporadic PPH. However, the genetic cause of PPH remains unclear in at least 45% of families. METHODS: We investigated 130 members of 10 families with at least 1 PPH patient, recruited without selection for familial disease. Manifest PPH was documented in 21 individuals. An increase in pulmonary artery systolic pressure (PASP) above 40 mm Hg during supine bicycle exercise was found in 46 healthy individuals. Their PASP increased from 21.0 +/- 4.6 mm Hg at rest to 54.0 +/- 9.8 mm Hg during exercise. In 51 relatives, PASP values were normal at rest and during exercise, and 12 members were classified as status unknown. RESULTS: Two families showed a mutation in the BMPR2 gene. Three families with no BMBR2 mutation showed evidence for linkage to a more proximal location on chromosome 2q31 (odds ratio [OR] for linkage 1.1.10(6):1). This locus, designated PPH2, maps in-between the markers D2S335 and D2S2314. We obtained significant support for heterogeneity in PPH with an OR of 2.8.10(11). CONCLUSIONS: We conclude that PPH may be a genetically heterogeneous disorder with at least two-and possibly more-causative genes.

Adolescent↗