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At least 163 records · Page 9Linked to original sources
PubMed2006

Integrity of the Mod(mdg4)-67.2 BTB domain is critical to insulator function in Drosophila melanogaster.

The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

Alleles↗
PubMed2005

Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes.

Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo.

Animals↗
PubMed2002

Chromatin structure of eukaryotic promoters: a changing perspective.

Over the past few years, many studies have attempted to determine the role of nucleosomes as both positive and negative transcription regulators. The emphasis has mostly centered on chromatin remodeling activities and histone modifications, leaving the question of the influence of the higher-order structure out of the spotlight. Recent technical developments allowing direct measurements of size and mechanical properties of in vivo assembled chromatin may shed light on this poorly understood area. This article presents a brief summary of the current knowledge on transcription-dependent chromatin dynamics and how a rather simple agarose electrophoresis method may change the current view on structural changes linked to transcriptional activation of chromatin.

Animals↗
PubMed2003

The many roles of the transcriptional regulator CTCF.

The nuclear factor CTCF was first identified as one of the factors binding to the regulatory regions of the c-myc gene. Further study of this protein revealed roles in transcriptional repression, insulator function, and imprinting genetic information. Recent studies have provided new insight into the mechanism through which this factor acts at various levels of gene regulation.

Animals↗
PubMed2005

Successful correction of the human Cooley's anemia beta-thalassemia major phenotype using a lentiviral vector flanked by the chicken hypersensitive site 4 chromatin insulator.

beta-Thalassemias are the most common single-gene disorders and are potentially amenable to gene therapy. While retroviral vectors carrying the human beta-globin cassette were notoriously unstable and expressed poorly, considerable progress has now been made using lentiviral vectors (LVs), which stably transmit the beta-globin expression cassette. Mouse studies using LVs have shown correction of the beta-thalassemia-intermedia phenotype and a partial, variable correction of the mouse beta-thalassemia major phenotype, despite the use of beta-globin-hypersensitive sites that are known to result in position-independent effects. Our group used the alpha-globin-hypersensitive site in self-inactivating (SIN) LVs with long-term expression in secondary mice that resisted methylation-associated proviral silencing. However, these vectors also suffered from chromatin position effects. We therefore flanked a SIN-lentiviral vector carrying the human beta-globin expression cassette with a chromatin insulator and studied expression in bone marrow from four patients with transfusion-dependent human thalassemia major. We demonstrated normal levels of human beta-globin expression in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3-4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal control subjects. Results show genetic correction of primitive human progenitor cells and normalization of the human thalassemia major phenotype.

Animals↗
PubMed2004

Nuclear location of a chromatin insulator in Drosophila melanogaster.

Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.

Animals↗
PubMed2006

A look to future directions in gene therapy research for monogenic diseases.

The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise.

Animals↗
PubMed2004

Preparation of recombinant proteins in milk.

Using transgenic animals as the source of recombinant proteins has several specific advantages. Large amounts of proteins can be obtained, essentially from milk. These proteins are often properly processed. They are in a number of cases correctly folded, assembled, cleaved, glycosylated, gamma-carboxylated, and so on. Purification of recombinant proteins from milk is not a particularly difficult task. The level of expression of foreign genes in milk cannot be predicted in all cases and appropriate vectors must be used. Generation of transgenic mice is popular but their production is quite limited. Transgenesis in larger animals, rabbits and farm animals, is achieved essentially by a few companies. Some recombinant proteins may be found in blood circulation and alter animal health. Milk from transgenic animals has become a quite attractive alternative to other sources of recombinant proteins.

Animals↗
PubMed2005

Retrovirus silencing and vector design: relevance to normal and cancer stem cells?

An obstacle confronting gene therapy in stem cells is transcriptional silencing of the vector. Here, we discuss recent data indicating that oncoretrovirus and lentivirus vectors are silenced by multiple epigenetic pathways that result in DNA methylation and histone modifications. Both vector types can be variegated in stem cells and expression is often extinguished during differentiation. We propose a novel model of retrovirus silencing in which epigenetic pathways compete to recruit histone deacetylases, de novo methyltransferases, histone H1 and MeCP2 to the provirus. These chromatin modifications may act in concert with heterochromatin at or near the integration site to establish silencing or variegation respectively. Retrovirus vector designs for stem cells should delete virus silencer elements, incorporate strong positive regulatory elements and insulators, and avoid non-mammalian reporter genes. In addition, cancer stem cells that continually repopulate a growing tumour may share silencing pathways with normal stem cells. Ultimately, optimized vector designs may prove to be valuable tools for gene therapy of both normal and cancer stem cells.

Epigenesis, Genetic↗
PubMed2001

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

3T3 Cells↗
PubMedDate not supplied

Molecular basis of RhD-positive/D-negative chimerism in two patients.

This study investigated two patients with Rh chimerism: patient A, a healthy individual, and patient B with myelofibrosis. Flow cytometry studies showed two red blood cell populations of Rh phenotypes R1r and rr at percentages of about 25% and 75% respectively. Normal RhD transcript sequences were found following RT-PCR. Genomic DNA (gDNA) showed normal exon, intron, GATA regions and exon/intron boundary sequences except for a single base change in intron 7 (C --> A) of exon 7 in patient A. The major change found in both patients was the absence of RHD exon 9 DNA in gDNA isolated from peripheral blood. These findings suggest a somatic mutation, probably in a stem cell common to the myeloid lineage of both patients, and indicate that patient A may undergo malignant transformation in the future.

Adult↗
PubMed2006

[Insulators of higher eukaryotes: properties, mechanisms of action, and role in transcriptional regulation].

One of the dogmas of transcriptional regulation in higher eukaryotes suggests the existence of transcriptional domains with no promoter-enhancer interactions between them. Specific regulatory elements, known as insulators, block the interaction between an enhancer and a promoter. Insulators are believed to act as transcription domain boundaries. The review considers general properties of well-known insulators identified in Drosophila and vertebrates. The mechanism of action of insulators and their role in the regulation of gene expression are discussed on the basis of available information.

Animals↗
PubMed2002

Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex.

The extensive infraabdominal (iab) region contains a number of cis-regulatory elements, including enhancers, silencers, and insulators responsible for directing the developmental expression of the abdominal-A and Abdominal-B homeotic genes at the Drosophila bithorax complex. It is unclear how these regulatory elements are primed for activity early in embryogenesis, but the 100-kb intergenic region is subject to a complex transcriptional program. Here, we use molecular and genetic methods to examine the functional activity of the RNAs produced from this region and their role in cis regulation. We show that a subset of these transcripts demonstrates a distinct pattern of cellular localization. Furthermore, the transcripts from each iab region are discrete and the transcripts do not spread across the insulator elements that delineate the iab regions. In embryos carrying a Mcp deletion, the intergenic transcription pattern is disrupted in the iab4 region and the fourth abdominal segment is transformed into the fifth. We propose that intergenic transcription is required early in embryogenesis to initiate the activation of the Drosophila bithorax complex and define the domains of activity for the iab cis-regulatory elements. We also discuss a possible mechanism by which this may occur.

Animals↗
PubMed2004

The 5'-HS4 chicken beta-globin insulator is a CTCF-dependent nuclear matrix-associated element.

The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking insulators, of which the first example was found in a DNA sequence element, HS4, at the 5' end of the chicken beta-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 insulator element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the insulator is a distinct matrix-associated element.

Animals↗
PubMed2006

Unraveling cis-regulatory mechanisms at the abdominal-A and Abdominal-B genes in the Drosophila bithorax complex.

Genome sequencing has revealed that in metazoans, only a small percentage of DNA actually codes for functional proteins. Research efforts have focused on elucidating the purpose of the rest of the genome, which was initially largely thought of as mere 'junk' DNA. One genomic region that is proving to be a rich source of new information is the Drosophila bithorax complex (BX-C). At this homeotic gene complex, many different classes of cis-regulatory elements, such as insulators, silencers, enhancers, and promoters, work together to tightly control gene expression during development. Recent studies have begun to unravel the intricate nature of these regulatory interactions. The BX-C was first discovered and characterized by Ed Lewis over three decades ago. In his seminal 1978 Nature paper, Lewis speculated that "substances" originating from the nongenic regions of the BX-C may regulate expression of the neighboring abdominal-A and Abdominal-B homeotic genes. A number of discoveries in the last few years suggest that he was right. The activation of some of the cis-sequences at the complex appears to be controlled by nongenic transcription, providing a further level of regulatory complexity to regions of nonprotein coding DNA. The hope is that these studies of gene regulation at the BX-C in the humble fruit fly will provide clues as to how vast intergenic regions contribute to the incredible complexity of gene regulation in other species, including humans.

Animals↗
PubMed2005

The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator.

Chromatin insulators are gene regulatory elements implicated in the establishment of independent chromatin domains. The gypsy insulator of D. melanogaster confers its activity through a protein complex that consists of three known components, Su(Hw), Mod(mdg4)2.2, and CP190. We have identified a factor, Drosophila Topoisomerase I-interacting RS protein (dTopors) that interacts with the insulator protein complex and is required for gypsy insulator function. In the absence of Mod(mdg4)2.2, nuclear clustering of insulator complexes is disrupted and insulator activity is compromised. Overexpression of dTopors in the mod(mdg4)2.2 null mutant rescues insulator activity and restores the formation of nuclear insulator bodies. dTopors associates with the nuclear lamina, and mutations in lamin disrupt dTopors localization as well as nuclear organization and activity of the gypsy insulator. Thus, dTopors appears to be involved in the establishment of chromatin organization through its ability to mediate the association of insulator complexes with a fixed nuclear substrate.

Animals↗
PubMed2000

[Insulators and interaction between long-distance regulatory elements in higher eukaryotes].

Enhancers can activate a promoter located as far as several hundred kilobases away, which is specific for transcription regulation in higher eukaryotes. Notwithstanding the vast information accumulated on transcription regulation at various levels, the mechanism of long-distance enhancer-promoter interactions is still obscure. Modern views of insulators as elements modulating enhancer-promoter interactions are reviewed.

Enhancer Elements, Genetic↗