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LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

ABSTRACT: The DNaseI hypersensitive sites (HSs) of the human ?-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human ?-globin locus yeast artificial chromosome (?-YAC) lines were produced in which the ?-globin gene was replaced with a second marked ?-globin gene (?(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'?HS3) or a 5'HS3 core deletion (5'?HS3c). The 5'?HS3c mice expressed ?(m)-globin throughout development; ?-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type ?-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for ?(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ?-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except ?(m)-globin expression was higher during primitive erythropoiesis and ?-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

SUBMITTER: Peterson KR

PROVIDER: S-EPMC3526258 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

Peterson Kenneth R KR Fedosyuk Halyna H Harju-Baker Susanna S

Nucleic acids research 20121004 22

The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artifici ...[more]

PMID: 23042246

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Project description:The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

Dataset Information

LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

Publications

LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

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