Abstract
Acting in cis with the beta-globin locus control region, the CAAT box of the beta-globin gene promoter stimulates transcription 10-fold in murine erythroleukemia (MEL) cells but is without effect in K562 cells. Our previous studies suggested that of four proteins from MEL cells that bind to this CAAT box region (CP1, GATA-1, and two factors that were denoted DSFr and DSF1) DSFr is involved in the up-regulation of transcription. In the present report, the DSFr protein in MEL cells was identified as C/EBPgamma through expression cloning and antibody studies. C/EBPgamma DNA binding activity could not be detected in K562 cells. However, K562 cells, but not MEL cells, were found to express LIP, which is a truncated form of C/EBPbeta and is an inhibitor of transcription. Thus, the differential expression of C/EBP members could account for the ability of the beta-globin CAAT box to stimulate transcription in MEL cells, but not function in K562 cells. Juxtaposing a specific C/EBP binding sequence next to the beta-globin promoter, in constructs in which the CAAT box had been rendered inactive by mutation or deletion, restored full promoter activity in MEL cells only if CP1 still bound to the promoter. In conjunction with previous mutation analyses, these results suggest that C/EBPgamma may collaborate with CP1 to enhance transcription through the beta-globin CAAT box.
Highlights
The human -globin locus (Fig. 1) contains five active genes that are expressed in red blood cells at different times during development. ⑀-Globin is only expressed early in embryogenesis, the two ␥-globin genes are transcribed at high levels during fetal life, while the ␦- and -globin genes are expressed in late fetal life and throughout adult life
DSF1 and DSFr in murine erythroleukemia (MEL) Cells Are C/EBP and C/EBP␥, respectively—To clone the corresponding cDNAs for the DSF proteins, a gt11 expression library was screened with a DNA probe representing the CAAT box of the -globin promoter
In previous studies [15] we showed that whereas the wild type CAAT box of the -globin promoter can stimulate LCR enhanced transcription 10-fold in MEL cells, the CAAT box is completely inactive in K562 cells
Summary
The human -globin locus (Fig. 1) contains five active genes that are expressed in red blood cells at different times during development (reviewed in Ref. 1). ⑀-Globin is only expressed early in embryogenesis, the two ␥-globin genes are transcribed at high levels during fetal life, while the ␦- (a minor contributor) and -globin genes are expressed in late fetal life and throughout adult life. In conjunction with the TATA box, the CAAT, and the proximal CACC motifs can each stimulate activity 10-fold higher than the TATA box alone in MEL cells Both the CAAT and proximal CACC elements and the cognate transcription factors that bind to these sequences play an important role in regulating the -globin promoter in the presence of the LCR and, may be involved in LCR interactions. In these same studies it was found that the CAAT box, in cis with the LCR, was completely unable to increase transcription from the -globin promoter in K562 cells [15] It appeared that K562 cells did contain DSFr DNA binding activity. We sought to identify the DSFr and DSF1 proteins to be able to determine if there
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