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PubMed2002

Blockers and barriers to transcription: competing activities?

In the eukaryotic cell active and inactive genes reside adjacent to one another and are modulated by numerous regulatory elements. Insulator elements prevent the misregulation of adjacent genes by restricting the effects of the regulatory elements to specific domains. Enhancer blockers prevent enhancers from inadvertently activating neighboring genes, and recent results suggest that they might function by a conserved mechanism across species. These elements appear to disrupt enhancer-promoter "communications" by interacting with the regulatory elements and sequestering these elements into specific regions of the nucleus thus rendering them non-functional. Barrier elements insulate active genes from neighboring heterochromatin and recent results suggest that they function by specific localized recruitment of acetyltransferases that antagonize the spread of heterochromatin-associated deacetylases, thus preventing the propagation of heterochromatin.

Animals↗
PubMed2003

Increased in vitro and in vivo transgene expression levels mediated through cis-acting elements.

BACKGROUND: Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. METHODS: Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. RESULTS: Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. CONCLUSION: The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

Animals↗
PubMed2003

Rationally designed insulator-like elements can block enhancer action in vitro.

Insulators are DNA sequences that are likely to be involved in formation of chromatin domains, functional units of gene expression in eukaryotes. Insulators can form domain boundaries and block inappropriate action of regulatory elements (such as transcriptional enhancers) in eukaryotic nuclei. Using an in vitro system supporting enhancer action over a large distance, the enhancer-blocking insulator activity has been recapitulated in a highly purified system. The insulator-like element was constructed using a sequence-specific DNA-binding protein making stable DNA loops (lac repressor). The insulation was entirely dependent on formation of a DNA loop that topologically isolates the enhancer from the promoter. This rationally designed, inducible insulator-like element recapitulates many key properties of eukaryotic insulators observed in vivo. The data suggest novel mechanisms of enhancer and insulator action.

Base Sequence↗
PubMed2006

The CD20/alphaCD20 'suicide' system: novel vectors with improved safety and expression profiles and efficient elimination of CD20-transgenic T cells.

Adoptive transfer of T lymphocytes is an attractive strategy for many experimental treatment strategies for cancer. Unfortunately, manipulated T cells could be responsible for serious adverse events. Retroviral CD20-transduced T cells may be able to control these unwanted effects. CD20-positive cells are sensitive to rituximab (RTX), a monoclonal antibody specific for CD20. This permits their selective elimination in vivo in case of adverse events. To this end, a system is required that permits efficient and safe transduction of donor T cells and effective elimination of CD20-positive T cells. We constructed different CD20-encoding retroviral vectors and investigated the impact of inclusion of the woodchuck post-transcriptional regulatory element (WPRE) and the chicken hypersensitivity site 4 insulator elements on the levels, homogeneity and stability of CD20 expression. Importantly, inclusion of either WPRE or insulator elements in the retroviral vector resulted in a dramatic improvement in the stability of CD20 expression. The insulator element also led to a much more homogeneous level of CD20 expression. We also show the efficient elimination of the CD20-transgenic T cells via RTX by different effector mechanisms. In conclusion, we have constructed CD20-encoding retroviral vectors with improved efficiency and safety profiles, which can be used as a suicide strategy.

Adoptive Transfer↗
PubMedDate not supplied

Silencing of gene expression: implications for design of retrovirus vectors.

Transcriptional silencing of retroviruses poses a major obstacle to their use as gene therapy vectors. Silencing is most pronounced in stem cells which are desirable targets for therapeutic gene delivery. Many vector designs combat silencing through cis-modifications of retroviral vector sequences. These designs include mutations of known retroviral silencer elements, addition of positive regulatory elements and insulator elements to protect the transgene from negative position effects. Similar strategies are being applied to lentiviral vectors that readily infect non-dividing quiescent stem cells. Collectively these cis-modifications have significantly improved vector design but optimal expression may require additional intervention to escape completely the trans-factors that scan for foreign DNA, establish silencing in stem cells and maintain silencing in their progeny. Cytosine methylation of CpG sites was proposed to cause retroviral silencing over 20 years ago. However, several studies provide evidence that retrovirus silencing acts through methylase-independent mechanisms. We propose an alternative silencing mechanism initiated by a speculative stem cell-specific "somno-complex". Further understanding of retroviral silencing mechanisms will facilitate better gene therapy vector design and raise new strategies to block transcriptional silencing in transduced stem cells.

Chromatin↗
PubMed2006

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity.

Mouse telokin and SM22alpha promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22alpha promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22alpha transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22alpha transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells.

Animals↗
PubMed2004

Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster.

Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3' MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed.

Animals↗
PubMed1996

Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.

Animals↗
PubMed2005

Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF.

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 HP1gamma 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.

CCCTC-Binding Factor↗
PubMed2000

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences.

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, the tet activator (tTA) can still activate the promoter, even in the presence of sns, demonstrating that sns does not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that sns does not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within sns which are responsible for insulator activity, we have performed in vitro DNase I footprinting and EMSA analysis, and in vivo functional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that sns displays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by sns depends on the assembly of distinct DNA-protein complexes.

Animals↗
PubMed2001

Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci.

We have studied developmentally regulated patterns of histone acetylation at high resolution across approximately 54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken beta-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5' insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases.

5' Untranslated Regions↗
PubMed2003

The functional analysis of insulator interactions in the Drosophila embryo.

Chromatin boundaries or insulators modulate enhancer-promoter interactions in complex genetic loci. However, the mechanism underlying insulator activity is not known. Previous studies showed that the activity of the Drosophila suHw insulator is abolished by the tandem arrangement (pairing) of the insulator elements, suggesting that interactions between insulators or like elements may be involved in their enhancer-blocking mechanism. To test whether such phenomenon reflects a general property of chromatin insulators, we tested the effect of pairing on enhancer-blocking activity of 11 homologous and heterologous insulator combinations using suHw, scs, or SF1 insulators. We found that, unlike the homologous pairing of suHw, the heterologous combinations of suHw with other insulators do not reduce their enhancer-blocking activity. Rather, paired insulators exhibit a higher level of enhancer-blocking activity than either single insulator alone, suggesting that they can function independently or additively. Furthermore, the analyses of two additional chromatin boundaries, scs and SF1, in homologous or heterologous pairing with other boundary elements, also showed no reduction but rather enhancement of insulator activity. We propose that diverse mechanisms may underlie insulator activity, and selective interactions among insulators could influence their function as well as the formations of independent chromatin domains.

Animals↗
PubMed2006

A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities.

A previous study demonstrated that the latency-associated transcript (LAT) promoter and the LAT enhancer/reactivation critical region (rcr) are enriched in acetyl histone H3 (K9, K14) during herpes simplex virus type 1 (HSV-1) latency, whereas all lytic genes analyzed (ICP0, UL54, ICP4, and DNA polymerase) are not (N. J. Kubat, R. K. Tran, P. McAnany, and D. C. Bloom, J. Virol. 78:1139-1149, 2004). This suggests that the HSV-1 latent genome is organized into histone H3 (K9, K14) hyperacetylated and hypoacetylated regions corresponding to transcriptionally permissive and transcriptionally repressed chromatin domains, respectively. Such an organization implies that chromatin insulators, similar to those of cellular chromosomes, may separate distinct transcriptional domains of the HSV-1 latent genome. In the present study, we sought to identify cis elements that could partition the HSV-1 genome into distinct chromatin domains. Sequence analysis coupled with chromatin immunoprecipitation and luciferase reporter assays revealed that (i) the long and short repeats and the unique-short region of the HSV-1 genome contain clustered CTCF (CCCTC-binding factor) motifs, (ii) CTCF motif clusters similar to those in HSV-1 are conserved in other alphaherpesviruses, (iii) CTCF binds to these motifs on latent HSV-1 genomes in vivo, and (iv) a 1.5-kb region containing the CTCF motif cluster in the LAT region possesses insulator activities, specifically, enhancer blocking and silencing. The finding that CTCF, a cellular protein associated with chromatin insulators, binds to motifs on the latent genome and insulates the LAT enhancer suggests that CTCF may facilitate the formation of distinct chromatin boundaries during herpesvirus latency.

Animals↗
PubMed2001

CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus.

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.

Binding Sites↗
PubMed2005

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters.

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.

Animals↗
PubMed1995

Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.

The human apolipoprotein B (apoB) gene resides within a 47.5-kb chromatin domain that is flanked by sequences that bind to the nuclear matrix. These matrix attachment regions (MARs) are boundaries between nuclease-sensitive and -resistant chromatin. As domain boundaries are thought to function as insulator elements, shielding sequences between them from effects of neighboring chromatin, this raised the possibility that the apoB MARs have functions that could be assayed by transfection. To test this possibility, we examined effects of the apoB MARs on transgene expression in transiently and stably transfected rat and human hepatoma cells. The apoB MARs had no effects on expression of transiently transfected reporters, but they altered expression of stably integrated transgenes in dramatic and reproducible ways. Single integrated copies of transgenes that contained the apoB promoter and second intron enhancer, which are sufficient for high-level expression in transient assays, were expressed at low and variable levels in stable transfectant clones. In contrast, transgenes containing the apoB 5' and 3' MARs were expressed at levels nearly 200-fold higher than levels of the minimal reporters in stable transfectants, and expression was position independent. Transgenes that contained the apoB MARs and an additional 3.3 kb of apoB 5' flanking sequence were also expressed in an elevated, position-independent manner. Surprisingly, tandem transgene arrays in multicopy transfectants were transcriptionally inactive. These observations suggest that the apoB MARs function as insulator elements, shielding transgene expression from effects of neighboring chromatin domains.

Animals↗
PubMed2003

Replication of the chicken beta-globin locus: early-firing origins at the 5' HS4 insulator and the rho- and betaA-globin genes show opposite epigenetic modifications.

Chromatin structure is believed to exert a strong effect on replication origin function. We have studied the replication of the chicken beta-globin locus, whose chromatin structure has been extensively characterized. This locus is delimited by hypersensitive sites (HSs) that mark the position of insulator elements. A stretch of condensed chromatin and another HS separate the beta-globin domain from an adjacent folate receptor (FR) gene. We demonstrate here that in erythroid cells that express the FR but not the globin genes, replication initiates at four sites within the beta-globin domain, one at the 5' HS4 insulator and the other three near the rho- and beta(A)-globin genes. Three origins consist of G+C-rich sequences enriched in CpG dinucleotides. The fourth origin is A+T rich. Together with previous work, these data reveal that the insulator origin has unmethylated CpGs, hyperacetylated histones H3 and H4, and lysine 4-methylated histone H3. In contrast, opposite modifications are observed at the other G+C-rich origins. We also show that the whole region, including the stretch of condensed chromatin, replicates early in S phase in these cells. Therefore, different early-firing origins within the same locus may have opposite patterns of epigenetic modifications. The role of insulator elements in DNA replication is discussed.

Animals↗
PubMed2005

Simultaneous sequence transfer into two independent locations of a reporter vector using MultiSite Gateway technology.

The bacteriophage lambda recombination system is increasingly used for recombinant DNA applications that involve the frequent transfer of sequences into and between shuttle and reporter vectors. This approach bypasses the need for restriction endonucleases or ligases and, as such, is easily scalable and automated. However this system has not yet been tested for the ability to support the simultaneous introduction of donor fragments into two separate target sites of a single reporter plasmid. This attribute would greatly facilitate studies of cis-regulatory elements that only function in specific combinations, such as a class of regulatory elements known as chromatin insulators. With the goal of facilitating a screen for chromatin insulators, we sought to determine whether the commercially available MultiSite Gateway Technology recombination system could be used to simultaneously insert candidate insulator elements into two separate locations of a functional reporter plasmid. We show that this application is both highly efficient and specific, generating the desired recombination products nearly three quarters of the time without disrupting the specificity of the reporter system. As such, these studies establish a novel application of the MultiSite Gateway Technology for the generation of recombinant reporter plasmids where the constituent elements function in a combinatorial fashion.

Bacteriophage lambda↗