Linker Scanning Mutagenesis of the 5′-Flanking Region of the Mouse β-Major-Globin Gene: Sequence Requirements for Transcription in Erythroid and Nonerythroid Cells

Abstract

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

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).…”

“…The promoter DHSs of globin and other erythroid-specific genes in erythroid cell nuclei extend some 150 to 250 bp upstream of the transcription initiation site (4,23,24,26,39) and contain conserved sequences such as the ATA (-30 bp), CCAAT (-70 bp), and CACC motifs which are essential for promoter function (6,11,32). Additional proximal promoter (-1 to -100 bp) transcription factor binding sites, such as GATA-1 (36) (also called NF-E1, GF1, EF1, or Eryfl [17,36,38,49]) and Spl (26), have also been described, and it is the interactions between these proximal promoter nucleoprotein complexes which presumably favor the formation of an active transcription initiation complex.…”

In vitro characterization of tissue-specific nuclear proteins preferentially bound to the mouse .beta.-globin gene during MEL cell terminal differentiation

“…Therefore, this system is a very suitable model to study the tissue-specific factors that are activated in order to promote the selectivity of transcription, leading to a particular type of terminal phenotypic expression. Cis-acting DNA sequences that control human, murine, and chicken /3-globin gene transcription have been analyzed by deletion, point mutation, and chimeric gene constructions, using mainly so far either in vivo assays of transient expression of introduced genes (Charnay et al, 1984(Charnay et al, ,1985Dierks et al, 1983;Myers et al, 1986;Chao et al, 1983), in vitro transcription systems (Hofer et al, 1982), or transgenic mice (Kollias et al, 1987). These are usually located at chromatin hypersensitive sites [for a review, see Galson and Housman (1988)].…”