Abstract
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.
Highlights
Human ␥- to -globin gene switching is coordinated by several regulatory sequences including the gene promoters and the locus control region (LCR).1 The LCR contains 5 DNaseI super-hypersensitive sites (HS1-HS5) located 6 to 26 kb upstream of the ⑀-globin gene in the -globin gene cluster [1, 2]
This work tests the roles of HS3 alone and in combination with HS2 in these competitive interactions and examines the possibility that these HS sites act in concert to enhance - and ␥-globin expression in a developmental stage-specific manner
We have demonstrated that HS2 and HS3 each contribute distinct properties to the -globin LCR
Summary
Preparation of the Constructs for the Stable Transfection Assays— The constructs used in the stable transfection assays are shown in Fig. 2 and are HS3␥, HS3, HS3HS2␥, HS3HS2, HS3HS2␥TATA, HS3HS2␥CACCC, and HS3HS2␥SSE. Those with HS2 contain a 1.9-kb KpnI-PvuII fragment (GenBankTM HUMHBB coordinates 7764 – 9653) normally located about 11 kb 5Ј of the human ⑀-globin gene. Sequencing experiments to determine the two major alternative start sites for ␥-globin mRNA in cells with the HS3HS2␥TATA constructs were performed using the fmol sequencing kit (Promega) and the above primer from the ␥-globin gene. Each lane represents the RNA from a pool of cells from a different transfection assay. Sum test, and all findings were judged to be significant at an ␣-level of Յ0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
