Hypersensitive site 4 of the human β globin locus control region

Abstract

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

“…The tandem GATA sequences from the HS4 HSFE were used in the test construct. Factor binding to these sites has also been well characterized (21,24), and both elements are 6/6-bp matches of the consensus GATA-1-binding sequence (45). The spacing between the NF-E2-and GATAbinding elements was the same as in the HS4 HSFE (Fig.…”

“…These bands comigrate with the two bands seen with the wild-type tandem GATA sites from HS4. These bands are due to single and double occupancy of the two sites (21,24). Both gel shift bands are competed by 200-fold excess unlabeled self-and wild-type HS4 GATA sequences.…”

“…Previous work on HS4 from our own laboratory and the Grosveld laboratory and from the Felsenfeld laboratory on a homologous region of the chicken 3Ј ␤-globin enhancer have demonstrated that, in addition to the in vitro DNase I footprints associated with the AP-1/NF-E2 and GATA sites, there is also a centrally located Sp1 binding site (21,24,47). This suggested that the Sp1 site might be the missing element for HS formation.…”

“…This suggested that the Sp1 site might be the missing element for HS formation. Each of the LCR HS core regions contains several Sp1 binding sites (6,7,21,24,26). Next we constructed artificial HSs containing the HS4 Sp1 element in the same location as it is found within the HSFE (Fig.…”

Transcriptional activation by hypersensitive site three of the human β-globin locus control region in murine erythroleukemia cells

“…We and others have previously shown that the activity of the LCR resides in these hypersensitive site (HS) regions and identified the core elements as 200-300 bp fragments [10][11][12][13][14]. There is strong evidence suggesting that each HS retains the essential properties of the LCR, although each site varies with regard to the level of transcriptional stimulation per gene and its developmental specificity [15,16].…”