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Enhancers and insulators are involved in the regulation of gene expression, but the basic underlying mechanisms of action of these elements are unknown. We analyzed the individual effects of the enhancer and the insulator from Drosophila mobile elements copia [enh(copia)] and gypsy using transfected genetic constructs in S2 cells. This system excludes the influence of genomic cis regulatory elements. The enhancer-induced synthesis of 350-1050-nt-long enhancer RNAs (eRNAs) and H3K4me3 and H3K18ac marks, mainly in the region located about 300bp downstream of the enhancer. Insertion of the insulator between the enhancer and the promoter reduced these effects. We also observed the binding of dCTCF to the enhancer and to gypsy insulator. Our data indicate that a single gypsy insulator interacts with both the enhancer and the promoter, while two copies of the gypsy insulator preferentially interact with each other. Our results suggest the formation of chromatin loops that are shaped by the enhancer and the insulator.

The genomic organization into active and inactive chromatin domains imposes specific requirements for having domain boundaries to prohibit interference between the opposing activities of neighbouring domains. These boundaries provide an insulator function by binding architectural proteins that mediate long-range interactions. Among these, CTCF plays a prominent role in establishing chromatin loops (between pairs of CTCF binding sites) through recruiting cohesin. CTCF-mediated long-range interactions are integral for a multitude of topological features of interphase chromatin, such as the formation of topologically associated domains, domain insulation, enhancer blocking and even enhancer function.

Hox gene expression imparts segment identity to body structures along the anterior-posterior axis and is tightly governed by higher order chromatin mechanisms. Chromatin regulatory features of the homeotic complex are best defined in Drosophila melanogaster, where multiple cis-regulatory elements have been identified that ensure collinear Hox gene expression patterns in accordance with their genomic organization. Recent studies focused on delineating the epigenetic features of the vertebrate Hox clusters have helped reveal their dynamic chromatin organization and its impact on gene expression. Enrichment for the 'activating' H3K4me3 and 'repressive' H3K27me3 histone modifications is a particularly strong read-out for transcriptional status and correlates well with the evidence for chromatin loop domain structures and stage specific topological changes at these loci. However, it is not clear how such distinct domains are imposed and regulated independent of each other. Comparative analysis of the chromatin structure and organization of the homeotic gene clusters in fly and mammals is increasingly revealing the functional conservation of chromatin mediated mechanisms. Here we discuss the case for interspersed boundary elements existing within mammalian Hox clusters along with their possible roles and mechanisms of action. Recent studies suggest a role for factors other than the well characterized vertebrate boundary factor CTCF, such as the GAGA binding factor (GAF), in maintaining chromatin domains at the Hox loci. We also present data demonstrating how such regulatory elements may be involved in organizing higher order structure and demarcating active domains of gene expression at the mammalian Hox clusters.

In the bithorax complex of Drosophila melanogaster, the chromatin boundary elements (BE) demarcate cis-regulatory domains that regulate Hox genes along the anteroposterior body axis. These elements are closely associated with the Polycomb Response Elements (PREs) and restrict the ectopic activation of cis-regulatory domains during development. The relevance of such specific genomic arrangements of regulatory elements remains unclear. Deletions of individual BE-PRE combination result in distinct homeotic phenotypes. In this study, we show that deletion of two such BE-PRE combinations in cis leads to new genetic interactions, which manifests as dorsal closure defect phenotype in adult abdominal epithelia. We further demonstrate that dorsal closure phenotype results from enhanced and ectopic expression of Hox gene Abd-B in the larval epithelial cells. This suggests a specific role of multiple BE-PRE combinations in the larval epithelial cells for regulation of Abd-B. Using chromosome conformation capture experiments, we show that genetic interactions correlate with direct physical interactions among the BE-PRE combinations. Our results demonstrate the functional relevance of the closely associated BE and PRE combinations in regulation of Hox genes.

Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

The class IV alcohol dehydrogenase gene ADH7 encodes an enzyme that is involved in ethanol and retinol metabolism. ADH7 is expressed mainly in the upper gastrointestinal tract and not in the liver, the major site of expression of the other closely related ADHs. We identified an intergenic sequence (iA1C), located between ADH7 and ADH1C, that has enhancer-blocking activity in liver-derived HepG2 cells that do not express their endogenous ADH7. This enhancer blocking function was cell- and position-dependent, with no activity seen in CP-A esophageal cells that express ADH7 endogenously. iA1C function was not specific to the ADH enhancers; it had a similar cell-specific effect on the SV40 enhancer. The CCCTC-binding factor (CTCF), an insulator binding protein, bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of ADH enhancers and thereby contributes to the cell specificity of ADH7 expression.

Epigenetic mechanisms play a fundamental role in generating diverse and heritable patterns of viral and cellular gene expression. Epstein–Barr virus (EBV) can adopt a variety of gene expression programs that are necessary for long-term viral persistence and latency in multiple host-cell types and conditions. The latent viral genomes assemble into chromatin structures with different histone and DNA modifications patterns that control viral gene expression. Variations in nucleosome organization and chromatin conformations can also influence gene expression by coordinating physical interactions between different regulatory elements. The viral-encoded and host-cell factors that control these epigenetic features are beginning to be understood at the genome-wide level. These epigenetic regulators can also influence viral pathogenesis by expanding tissue tropism, evading immune detection, and driving host-cell carcinogenesis. Here, we review some of the recent findings and perspectives on how the EBV epigenome plays a central role in viral latency and viral-associated carcinogenesis.

International audience; Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks'' of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.

Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed.

A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome.

tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer - blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols.

The Dual Reciprocity Method is a popular mathematical technique to treat domain integrals in the boundary element method (BEM). This technique has been used to treat inertial integrals in the dynamic thin plate bending analysis using a direct formulation of the BEM based on the elastostatic fundamental solution of the problem. In this work, this approach was applied for the dynamic analysis of shear deformable plates based on the Reissner plate bending theory, considering the rotary inertia of the plate. Three kinds of problems: modal, harmonic and transient dynamic analysis, were analyzed. Numerical examples are presented to demonstrate the efficiency and accuracy of the proposed formulation. © 2011 Elsevier Ltd. All rights reserved

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.

In this study, we have constructed the first organic field effect sensor based on an electrolyte-insulator-semiconductor structure (OEIS) and applied this novel device to pH and DNA sensing. Variations in the insulator-electrolyte surface potential, which originate from either the change of the ionization states of the insulator surface groups or the binding of charged molecules to the insulator surface, modify the flat band voltage (V(FB)) of the OEIS sensor. The pH sensing experiments of OEIS sensor showed that the output signal linearly depended on pH solution in the range from pH 2 to pH 12, and an average sensitivity of 44.1 mV/pH was obtained. In the biosensing experiments, the absorption of positively charged poly-L-lysine on the insulator surface resulted in the reduction of the V(FB) value, whereas the subsequent binding of negatively charged single-stranded DNA probe (ssDNA) via electrostatic interaction increased the V(FB) value. Furthermore, the ssDNA-immobilized OEIS device was successfully used for the detection of DNA hybridization. The detection limit of complementary DNA was as low as 1 microM, and the output signal of OEIS biosensor linearly increased with the logarithm of complementary DNA concentration in the range from 5x10(-5) to 10(-7) M. The easy and inexpensive fabrication of the OEIS device allows to be served as a potentially disposable and sensitive biosensor.

The homeotic genes are essential to the patterning of the anterior-posterior axis along the developing Drosophila embryo. The expression timing and levels of these genes are crucial for the correct specification of segmental identity. The Abdominal-B (Abd-B) gene is first detected in the most posterior abdominal segments at high levels and gradually appears in progressively anterior abdominal segments in lower amounts. Regulatory mutations affecting this expression pattern produce homeotic transformations in the abdomen. The promoter targeting sequences (PTS) from Abd-B locus overcome the enhancer blocking effect of insulators and facilitate long-range enhancer–promoter interactions in transgenic flies (1, 2). In this study, we found that transgene activation by the IAB5 enhancer can be delayed by inserting a 9.5 kb 3′ Abd-B regulatory region containing the Frontabdominal-8 (Fab-8) insulator and the PTS element. We found that the delay is caused by the PTS and an insulator, and it is not specific to the enhancer or the promoter tested. Based on these findings, we hypothesize that the delay of remote enhancers is responsible for the Abd-B expression pattern, which is at least in part due to the regulatory activities of the PTS elements and chromatin boundaries.

Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation.

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken beta-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.

The retinoid X receptor beta gene (Rxrb) is located just upstream of the alpha2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF.

Summary Paenibacillus polymyxa PKB1 produces fusaricidins, a family of lipopeptide antibiotics that strongly inhibits the growth of many plant pathogenic fungi. The fusaricidin biosynthetic gene cluster was cloned and sequenced, and it spans 32.4 kb, including an open reading frame ( fusA ) encoding a six-module nonribosomal peptide synthetase. The second, fourth, and fifth modules of fusaricidin synthetase each contain an epimerization domain, consistent with the structure of fusaricidins. However, no epimerization domain is found in the sixth module, corresponding to d-Ala. This sixth adenylation domain was produced at a high level in Escherichia coli and is shown to activate d-Ala specifically, providing evidence for direct activation of a d-amino acid by a prokaryotic peptide synthetase. The fusaricidin gene cluster also includes genes involved in the biosynthesis of the lipid moiety, but no genes for resistance, regulation, or transport functions were encountered.

CTCF plays a central role in vertebrate insulators and forms part of the Fab-8 insulator in Drosophila. dCTCF is present at hundreds of sites in the Drosophila genome, where it is located at the boundaries between bands and interbands in polytene chromosomes. dCTCF colocalizes with CP190, which is required for proper binding of dCTCF to chromatin, but not with the other gypsy insulator proteins Su(Hw) or Mod(mdg4)2.2. Mutations in the CP190 gene affect Fab-8 insulator activity, suggesting that CP190 is an essential component of both gypsy and dCTCF insulators. dCTCF is present at specific nuclear locations, forming large insulator bodies that overlap with those formed by Su(Hw), Mod(mdg4)2.2, and CP190. The results suggest that Su(Hw) and dCTCF may be the DNA-binding components of two different subsets of insulators that share CP190 and cooperate in the formation of insulator bodies to regulate the organization of the chromatin fiber in the nucleus.

Retroviral vectors have been developed for gene therapy of blood disorders because they achieve long-term expression of the transgene. However, interactions between the regulatory elements contained in such vectors and cellular genes may result in pathogenic proto-oncogene activation. We designed a cassette consisting of a splice acceptor followed by the coding sequences for Green Fluorescent Protein (GFP) and a polyadenylation site which was inserted in a reverse orientation in self-inactivating lentiviral vectors. Retroviral vectors integrate preferentially in genes and in one-half of the integrations, the expression cassette will be in a tandem orientation with respect to the gene's promoter and potentially expressed when the trapped promoter is intrinsically active or activated by regulatory elements within the vector. Approximately 10% of the integrations of the control vector with only the GFP cassette resulted in expression of the GFP marker. The trapping cassette in vectors containing globin regulatory elements was expressed more frequently in erythroleukemia (K562) cells than the control cassette only vector but there was no increase in promoter trapping efficiency in HeLa cells. In contrast, we found that addition of a gammaretroviral long terminal repeat increased the frequency of GFP expression both in K562 and HeLa cells. Promoter activation in K562 cells by vectors containing globin regulatory elements was significantly reduced by addition of flanking insulator elements.

Insulator elements can be classified as enhancer-blocking or barrier insulators depending on whether they interfere with enhancer-promoter interactions or act as barriers against the spreading of heterochromatin. The former class may exert its function at least in part by attaching the chromatin fiber to a nuclear substrate such as the nuclear matrix, resulting in the formation of chromatin loops. The latter class functions by recruiting histone modifying enzymes, although some barrier insulators have also been shown to create chromatin loops. These loops may correspond to functional nuclear domains containing clusters of co-expressed genes. Thus, insulators may determine specific patterns of nuclear organization that are important in establishing specific programs of gene expression during cell differentiation and development.

When placed between an enhancer and promoter, certain DNA sequence elements inhibit enhancer-stimulated gene expression. The best characterized of these enhancer-blocking insulators, gypsy in Drosophila and the CTCF-binding element in vertebrates and flies, stabilize contacts between distant genomic regulatory sites leading to the formation of loop domains. Current results show that CTCF mediates long-range contacts in the mouse beta-globin locus and at the Igf2/H19-imprinted locus. Recently described active chromatin hubs and transcription factories also involve long-range interactions; it is likely that CTCF interferes with their formation when acting as an insulator. The properties of CTCF, and its newly described genomic distribution, suggest that it may play an important role in large-scale nuclear architecture, perhaps mediated by the co-factors with which it interacts in vivo.

Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.

The tandemly repeated sea urchin alpha-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the alpha-H2A gene depends on the binding of the MBF-1 activator to the 5' enhancer, while down-regulation relies on the functional interaction between the 3' sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to up-regulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3' insulator and 5' regulatory regions, in the repression of the alpha-H2A gene despite the presence of the MBF-1 activator bound to the enhancer.

Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.

Chromatin insulators are boundary elements between distinctly regulated, neighboring chromosomal domains, and they function by blocking the effects of nearby enhancers in a position-dependent manner. Here, we show that the SNF2-like chromodomain helicase protein CHD8 interacts with the insulator binding protein CTCF. Chromatin immunoprecipitation analysis revealed that CHD8 was present at known CTCF target sites, such as the differentially methylated region (DMR) of H19, the locus control region of beta-globin, and the promoter region of BRCA1 and c-myc genes. RNA interference-mediated knockdown of CHD8 significantly abolished the H19 DMR insulator activity that depends highly on CTCF, leading to reactivation of imprinted IGF2 from chromosome of maternal origin. Further, the lack of CHD8 affected CpG methylation and histone acetylation around the CTCF binding sites, adjacent to heterochromatin, of BRCA1 and c-myc genes. These findings provide insight into the role of CTCF-CHD8 complex in insulation and epigenetic regulation at active insulator sites.

Chromatin insulators have been shown to stabilize transgene expression. Although insulators have been suggested to regulate the subcellular localization of chromosomes, it is still unclear whether this property is important for their anti-silencing activity. To investigate the underlying mechanisms governing the anti-silencing function of insulators, we studied the association of sea urchin arylsulfatase insulator (ArsI) with the nuclear matrix, which is a key component of the subnuclear localization of the genome. ArsI did not potentiate the nuclear matrix association with the transgene, even though it showed strong anti-silencing activity. This observation was in clear contrast to the results of the experiment using a human interferon-beta scaffold attachment region, in which the anti-silencing effect coincided with the enhanced matrix association. Chromatin immunoprecipitation analyses suggested that the absence of the matrix binding by ArsI was due to a lack of its binding to CCCTC-binding factor (CTCF), a protein known to be associated with matrix binding by chicken beta-globin insulator. Furthermore, ArsI maintained the nucleosome occupancy within the transgene at a constant level during long-term culture, although ArsI itself was not a nucleosome-excluding sequence. Taken together, these results suggest that this insulator exerts its anti-silencing activity by counteracting silencing-associated factors to maintain local chromatin environment, rather than by remodeling the subnuclear localization of the transgene locus.

Summary Prior studies of the DM1 locus have shown that the CTG repeats are a component of a CTCF-dependent insulator element and that repeat expansion results in conversion of the region to heterochromatin. We now show that the DM1 insulator is maintained in a local heterochromatin context: an antisense transcript emanating from the adjacent SIX5 regulatory region extends into the insulator element and is converted into 21 nucleotide (nt) fragments with associated regional histone H3 lysine 9 (H3-K9) methylation and HP1γ recruitment that is embedded within a region of euchromatin-associated H3 lysine 4 (H3-K4) methylation. CTCF restricts the extent of the antisense RNA at the wild-type (wt) DM1 locus and constrains the H3-K9 methylation to the nucleosome associated with the CTG repeat, whereas the expanded allele in congenital DM1 is associated with loss of CTCF binding, spread of heterochromatin, and regional CpG methylation.

TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.

Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox.

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

The nuclear architecture of the interphase nucleus is established by laying down an intricate three-dimensional framework of higher-order chromatin structure. This arrangement is essential for the integration of complex biological processes such as DNA replication, RNA processing, and transcription. Boundary or insulator elements are emerging as key players in the establishment and maintenance of this organization.

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.

The effects of a genetic insulator (chicken cHS4) on transgene expression in a positive feedback system using the GAL4-VP16 activator and luciferase reporter plasmids were examined. The introduction of cHS4 enhanced luciferase expression in mouse liver, indicating that this system would be useful for efficient transgene expression.

The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

CTCF plays diverse roles in the regulation of eukaryotic genes. A new study by Lefevre et al. in a recent issue of Molecular Cell reveals a novel mechanism in which noncoding RNA transcription and nucleosome repositioning evicts CTCF from a regulatory element to facilitate induction of a nearby gene.