The gamma-globin promoter has a major role in competitive inhibition of beta-globin gene expression in early erythroid development.

The human gamma-globin gene competitively inhibits beta-globin gene expression in early erythroid development. To identify the gamma-globin gene sequences required for this effect, transgenic mice and stable transfection analyses with constructs containing 5'HS2 from the locus control region, modified gamma-globin genes, and the beta-globin gene were used. The -136 to +56 region of the gamma-globin promoter is necessary for competitive inhibition, as the beta-globin gene was inappropriately expressed in mouse embryos and in K562 and HEL cells containing constructs in which this region was deleted. Independently, the -140 to +56 region of gamma-globin gene was not sufficient to inhibit beta-globin transcription in mouse embryos or in cultured cells. Competitive inhibition of beta-globin gene expression was observed in K562 and HEL cells having a gamma-globin gene with a -161 promoter. The data suggest that the -161 gamma-globin promoter, which includes the CACCC box, two CCAAT boxes, the stage selector element (SSE), and TATA box, has a major role in suppressing beta-globin transcription early in development. Proteins binding to these or other gamma-globin promoter elements may interact with those binding to the locus control region, consequently precluding beta-globin transcription.

The human gamma-globin TATA and CACCC elements have key, distinct roles in suppressing beta-globin gene expression in embryonic/fetal development.

The competition model of globin gene regulation states that the gamma-globin gene precludes expression of the beta-globin gene in early development by competing for the enhancing activity of the locus control region. The gamma-globin gene with a -161 promoter is sufficient for suppressing beta-globin gene expression, and the gamma-globin TATA and CACCC elements are necessary for this effect. In this work, stable transfection and transgenic mouse assays have been performed with constructs containing HS3 and HS2 from the locus control region, the gamma-globin gene with promoter mutation(s), and the beta-globin gene. The data indicate that the gamma-globin TATA and CACCC elements together have at least an additive effect on the beta/gamma-globin mRNA ratio in early erythroid cells, suggesting that the elements work coordinately to suppress beta-globin gene expression. The TATA and CACCC are the major gamma-globin promoter elements responsible for this effect. Transgenic mouse experiments indicate that the gamma-globin TATA element plays a role in gamma-globin expression and beta-globin suppression in the embryo and fetus; in contrast, the CACCC element has a stage-specific effect in the fetus. The results suggest that, as is true for the erythroid Krüppel-like factor (EKLF) and the beta-globin promoter CACCC, a protein(s) binds to the gamma-globin CACCC element to coordinate stage-specific gene expression.

The roles of 5'-HS2, 5'-HS3, and the gamma-globin TATA, CACCC, and stage selector elements in suppression of beta-globin expression in early development.

The roles of HS2 and HS3 from the human beta-globin locus control region and of the TATA, CACCC, and stage selector elements of the gamma-globin promoter, in competitive inhibition of beta-globin gene expression in early development, were tested using stable transfections of HEL and K562 cells. Cells with an HS3gamma beta construct demonstrate that HS3 exhibits enhancing activity, but compared with HS2, this site participates less consistently in the inhibition of embryonic/fetal beta-globin expression. In cells with HS3HS2gamma beta constructs, the two HS sites act in concert to more effectively enhance gamma-globin gene expression and to drive stage-specific expression of the gamma- and beta-globin genes. A gamma-globin gene with a -161 promoter can competitively inhibit beta-globin gene expression. HS3HS2gamma beta constructs were used to determine the effects of gamma-globin promoter mutations within this region on competition. The CACCC and TATA elements, but not the stage selector element, inhibit inappropriate embryonic/fetal stage expression of the beta-globin gene. The mutation in the gamma-globin TATA element results in the use of two major alternative transcription start sites. The data suggest that proteins binding to the gamma-globin CACCC and TATA elements interact with those binding to HS2 and/or HS3 to preclude beta-globin transcription in early development.