Man Beta-globin Gene Cluster By Dnase I Hypersensitive Site 5'hs5 Functional Studies

A novel class of regulatory sequences, known as insulators, has properties consistent with a role in the demarcation of autonomous transcriptional domains. Insulators are defined by two functional properties. First, insulators block the activity of enhancers and silencers when inserted between these regulatory elements and a promoter. In this way, insulators perform the critical function of restricting enhancer and silencer action to the appropriate target promoters. These effects are established without inactivation of the regulatory elements. Second, insulators protect gene expression from positive and negative effects of chromatin surrounding a gene or gene locus. Mechanisms involved in the protection of nearby chromatin effects may involve the direct blocking of regulatory elements or the restriction of the spread of silencing complexes commonly associated with repressive chromatin. Insulators have been identified in many eukaryotic genomes, suggesting that these elements have a conserved role in defining domains of gene expression.The best characterized insulator element in vertebrates is the insulator in the chicken β-globin locus. In this locus, a relatively sharp transition in both DNase I sensitivity and histone hyperacetylation is observed roughly coincident with the position of a DNase I hypersensitive site (HS), 5'HS4. A 1.2 kb DNA fragment spanning 5'HS4 displays all the properties of an insulator: in human cell lines it provides a directional block to enhancer action on a promoter and it is capable of protecting a gene from position effects in Drosophila. Recently, a binding site for the ubiquitous DNA-binding protein CTCF was identified in 5'HS4, and the CTCF site is both necessary and sufficient for the enhancer blocking property of the insulator. A CTCF-binding site is also responsible for the enhancer-blocking activity of the chicken 3'HS and other vertebrate insulators. There is much conservation of structure and function at the multigene β-globin locus in vertebrates. In erythroid cells, the region upstream of the genes is marked by a series of HS that comprise the locus control region (LCR), which can confer high-level, copy number-dependent, position-independent β-globin gene expression in erythroid cells of transgenic mice.Evidences for the enhancer-blocking role of human β-globin locus 5'HS5 have mainly based on enhancer blocking assay or colony assay, stable transfection assay, and transgenic studies. However, these methods result in integration at random chromosomal locations of an uncontrolled number of transgene copies that express at levels that generally cannot be predicted or reproduced with precision because of position effects. Also, expression of a reporter gene has been assayed with methods that analyze total expression from a tissue or an aliquot of transfected cells. In these 'bulk' assays, increased expression could result from a higher proportion of cells expressing the gene of interest, or a higher rate of transcription in all cells, or both.Recombinase-mediated cassette exchange (RMCE) was developed to perform site-specific chromosomal integration using either the Cre or the Flp recombinases. The...