Dataset Information

An Erythroid-Specific Chromatin Opening Element Increases ?-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors.

ABSTRACT: Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the ?-globin locus control region (LCR) which is able to independently open local chromatin structure in erythroid tissues. This element includes binding sites for GATA-1, NF-E2, EKLF and Sp-1 and is evolutionarily conserved. We constructed a series of MSCV-based vectors containing the ?-globin gene driven by a minimal ?-globin promoter with combinations of the HSFE and LCR derived enhancer elements. Pools of MEL clones containing integrated vectors were analyzed for chromatin structure and ?-globin gene expression. The HSFE increased the extent of nuclease sensitive chromatin over the promoters of the constructs. The most effective vector included tandem copies of the HSFE and produced a 5-fold increase in expression compared to the promoter alone. These results indicate that the HSFE is able to augment the opening of ?-globin promoter chromatin structure and significantly increase gene expression in the context of an integrated retroviral vector.

SUBMITTER: Nemeth MJ

PROVIDER: S-EPMC1456070 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Publications

An Erythroid-Specific Chromatin Opening Element Increases β-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors.

Nemeth Michael J MJ Lowrey Christopher H CH

Gene therapy & molecular biology 20041201 B

Gene therapy strategies requiring long-term high-level expression from integrated genes are currently limited by inconsistent levels of expression. This may be observed as variegated, silenced or position-dependent gene expression. Each of these phenomena involve suppressive chromatin structures. We hypothesized that by actively conferring an open chromatin structure on integrated vectors would increase transgene expression. To test this idea we used a 100bp element from the β-globin locus contr ...[more]

PMID: 16670777

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Project description:The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron.A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis.Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway.Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.

Dataset Information

An Erythroid-Specific Chromatin Opening Element Increases ?-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors.

Publications

An Erythroid-Specific Chromatin Opening Element Increases β-Globin Gene Expression from Integrated Retroviral Gene Transfer Vectors.

Similar Datasets

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