Maximal Activity of an Erythroid-specific Enhancer Requires the Presence of Specific Protein Binding Sites in Linked Promoters

Abstract

High level expression of many eukaryotic genes is achieved through the action of distal regulatory sequences or enhancers. We have utilized the interaction between the erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the human ␤-globin locus control region and the globin gene promoters as a model to elucidate the mechanisms governing promoter/enhancer interactions. HS2 contains a 400-base pair core element consisting of tandem AP1/NF-E2 motifs flanked by binding sites for multiple ubiquitous and erythroidspecific factors. We have compared the enhancer activity of this core element with a synthetic enhancer lacking the factor binding sites flanking the AP1/NF-E2 motif (HS2 M ). In fetal/erythroid K562 cells, enhancement of a linked ␥-promoter was significantly greater with wild-type HS2 than with HS2 M . In contrast, the increase in ␤-promoter activity in these cells was equivalent with either enhancer fragment. Truncation of the binding site for the fetal/erythroid-specific stage selector protein in the ␥-promoter abolished the additional enhancer activity of HS2. Similarly, insertion of the stage selector protein site into the ␤-promoter boosted enhancer activity observed with HS2 but not HS2 M . In adult erythroid MEL cells, enhancement of a linked ␤-promoter was significantly greater with HS2 than with HS2 M . This effect was dependent on the binding of the adult stage-specific factor, erythroid Kruppel-like factor, to the ␤-promoter. Taken together, this data suggests that the stage-specific factors binding the proximal globin promoters and the factors flanking the AP1/ NF-E2 motif of HS2 act in synergy.

UNDERSTANDING FETAL GLOBIN GENE EXPRESSION: A STEP TOWARDS EFFECTIVE HbF REACTIVATION IN HAEMOGLOBINOPATHIES

“…The trans ‐acting environment, in particular the stage‐specific factors SSP and EKLF, also play a critical role, conferring a competitive advantage on the γ‐ and β‐promoters respectively ( Jane et al , 1992 ; Wijgerde et al , 1996 ; Perkins et al , 1996 ). Although the mechanisms by which these factors recruit the LCR have not been established, one model with increasing experimental support suggests that it may be achieved through direct interactions between the stage‐specific factors and LCR‐binding proteins, and the co‐activators of the polymerase complex ( Amrolia et al , 1997 , 1998).…”

Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2

“…In chicken, cCP2 regulates the lens-specific expression of the aA-crystalline gene [14]. In mammals, CP2 contributes to the preferential recruitment of the b-globin locus control region to the c-globin promoter during fetal erythropoiesis [20][21][22][23][24][25]. CP2 participates in cell-cycle regulation by binding to and thus modulating transcription of the thymidylate synthase (TS) promoter in growth-stimulated human cells of late G 1 phase [26][27][28].…”

cAMP response element‐binding protein 1 is required for hydroxyurea‐mediated induction of γ‐globin expression in K562 cells

“…In contrast, inclusion of the HS2 region of the LCR next to the Aγ promoter (−301 to +35) appears to confer an HU response, as exemplified by induction of reporter activity in transient or stable assays . To this end, a 0.7 kb region of the HS2‐LCR, which has enhancer activity in K562 cells, was also positioned upstream of the γ‐globin promoters …”