Sequences Flanking Hypersensitive Sites of the β-Globin Locus Control Region Are Required for Synergistic Enhancement

Abstract

The major distal regulatory sequence for the ␤-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.Genes in the ␤-globin gene complex (HBBC, containing HBE1, HBG2, HBG1, HBD, and HBB), together with those in the ␣-globin gene complex (HBZ2, HBA2, and HBA1) encode the developmentally regulated, erythroid-specific family of hemoglobins in vertebrates. Transcription of the mammalian HBBC is regulated both by proximal elements, such as promoters, and by a distal regulatory element known as the locus control region (LCR). The LCR is marked by several hypersensitive sites (HSs) in erythroid chromatin (18,45) and is required for high-level expression of genes within the HBBC in erythroid cells (reviewed in references 6 and 19). Transfection and transgenic mouse studies show that the LCR confers this high-level expression at many, but perhaps not all, ectopic sites of integration (1, 21, 31). Gain-of-function experiments examining multiple integrated copies of LCR constructs revealed expression that is copy number dependent and independent of the site of integration (e.g., see references 43 and 44), suggesting that the LCR contains a dominant chromatin-opening activity. However, deletion of HS1 to HS6 from the LCR of mouse Hbbc (3, 12), as well as deletion of HS2 to HS5 from the LCR of human HBBC on chromosome 11 (37), leaves the globin genes in an open chromatin domain, albeit expressed at very low to undetectable levels. Thus, the LCR is clearly required for enhancement but it is not necessary fo...

Deletion of the core region of 5′ HS2 of the mouse β-globin locus control region reveals a distinct effect in comparison with human β-globin transgenes

“…18 If the LCR forms a holocomplex then it follows that disruption of some portion of the structure could lead to disruption of the entire structure. This model is supported by transfection studies of constructs with different HS combinations 13,19,20 and by deletion studies of human LCR HS core regions in human ␤-globin yeast artificial chromosome (YAC) transgenic mice. 18,[21][22][23] In YAC transgenic mice, deletion of individual human HS cores (HS2, HS3, or HS4) results in severe reduction of all ␤-like globin gene expression and failure to form the remaining HSs, 21,24,25 suggesting that LCR HSs are mutually interdependent for their formation.…”

“…The flanking sequences do form minor DNaseI HSs and carry limited transcriptional activation activities. 13 Two models can be delineated for how the multiple LCR HSs interact to regulate gene expression. An additive model proposes that each 5Ј HS contributes independently in an additive manner to LCR activity.…”

Dynamic GATA Factor Interplay at a Multicomponent Regulatory Region of the GATA-2 Locus

“…Deletion of individual HSs of the endogenous ␤-globin locus control region yielded incremental decreases in ␤-globin expression, consistent with a mechanism involving additivity (46,47). In contrast, deletion of HSs from the intact ␤-globin locus within a yeast artificial chromosome (48,49) and transfection experiments (50,51) reveal synergism between the HSs.…”

A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human β-globin gene transfer

“…Globin transcriptional control elements have been extensively studied, 27,28 without conclusively defining what minimal segments and what combination thereof are best suited to generate a miniature ␤-globin transcription unit. The relative roles of the locus control region and of other genetic elements spanning the human ␤-globin locus and its chromatin domain in regulating globin gene expression remain controversial.…”