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USF binding sequences from the HS4 insulator element impose early replication timing on a vertebrate replicator.

ABSTRACT: The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the ?-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.

SUBMITTER: Hassan-Zadeh V

PROVIDER: S-EPMC3295818 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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USF binding sequences from the HS4 insulator element impose early replication timing on a vertebrate replicator.

Hassan-Zadeh Vahideh V Chilaka Sabarinadh S Cadoret Jean-Charles JC Ma Meiji Kit-Wan MK Boggetto Nicole N West Adam G AG Prioleau Marie-Noëlle MN

PLoS biology 20120306 3

The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested ...[more]

PMID: 22412349

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Project description:Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value.

Dataset Information

USF binding sequences from the HS4 insulator element impose early replication timing on a vertebrate replicator.

Publications

USF binding sequences from the HS4 insulator element impose early replication timing on a vertebrate replicator.

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