A Directly Repeated Sequence in the β-Globin Promoter Regulates Transcription in Murine Erythroleukemia Cells

Abstract

We have identified a previously undetected cis-acting element in the mouse beta-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the beta-globin direct-repeat element (beta DRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The beta DRE motif is highly conserved in all adult mammalian beta-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the beta DRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the beta DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in beta DRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the beta DRE have implications for the mechanism of action of the element in regulating beta-globin transcription and for mutational studies of other repetitive or redundant transcription elements.

Derepression of Mouse β-Major-Globin Gene Transcription during Erythroid Differentiation

“…5). The proximal GATA-1 (pPG60M) and CACC (p,CCM) promoter mutants are comparatively inactive, confirming and extending previous results (6,11,32,44). In contrast, the distal NF1 (pPNFlM), GATA-1 (pPi215M), and BB1 (pPBBlM) promoter mutants are fourto eightfold more active than ppWT.…”

“…The nuclear DHS of the mouse ,-major-globin gene promoter in MEL cells spans a 250-bp (-1 to -250 bp) sequence (4). A GATA-1 protein binding site at -212 bp (38) and conserved motifs such as the CCAAT, CACC, ,BDRE, and TATA motifs, which were identified in functional assays (6,11,32,44), have been described. Using in vitro protein binding assays and competitor oligonucleotides (Fig.…”

“…These regulatory elements reside in developmentally regulated and tissue-specific nuclear DNase I-hypersensitive sites (DHSs) (4,23,24,26,37,39,47) and are accessible for binding transcription factors in vivo (23,24,26,39). The characterization of promoter sequences in the absence of enhancer or LCR elements has defined transcription factor binding sites which are required for the modulation of both promoter activity and promoter function (5,6,11,12,14,18,23,32,38,44) and which may mediate putative promoter-enhancer-LCR interactions (1,2,7,8,10,16,28,29,35,48,50).…”

Engineering of the endogenous HBD promoter increases HbA2

“…While Pol II is a binding partner of TFIIB in the pre-initiation complex, this ChIP does have the limitation of indirectly immunoprecipitating other factors in the transcriptional pre-initiation complex and might also reflect general promoter activity. The β-DRF motif has been shown to be important for high transcription of HBB , but its bona fide binding factor has not yet been identified Stuve and Myers, 1990 . In the homozygous clones K and KDT, which harbor the edited KLF1 sequence, we observed increased binding of KLF1 to the HBD promoter relative to unedited WT HUDEP-2 cells.…”