Activation of the beta-globin promoter by the locus control region correlates with binding of a novel factor to the CAAT box in murine erythroleukemia cells but not in K562 cells.
Four distinct factors in extracts from murine erythroleukemia (MEL) cells interacted with the human beta-globin gene promoter CAAT box: CP1, GATA-1, and two novel factors, denoted a and b, one of which is highly inducible in the MEL system. GATA-1 binding to the CAAT element was very unstable (half-life < 1 min), whereas bindings of a, b, and CP1 were comparatively stable, with half-lives of 18, 19, and 3.5 min, respectively. Stable transfections of MEL cells showed that in the presence of the beta-globin locus control region (LCR), the wild-type CAAT box, a mutant which bound to GATA-1 with increased stability over the normal sequences, and a mutant which bound a, b, and CP1 specifically could all stimulate transcription greater than ninefold over that induced by a null CAAT mutation in both uninduced and terminally differentiated MEL cells. A mutant which bound the a and b factors specifically gave only a twofold stimulation of promoter activity, and this lower activity correlated with a decrease in the stability of binding of the b protein. On the other hand, CP1 binding alone did not stimulate transcription. Taken together, these results suggest that in the context of the wild-type beta-globin CAAT element the b factor stimulates transcription directed by the LCR in MEL cells, although the LCR can also function through more stable GATA-1-binding sequences. However, in K562 cells, the wild-type beta-globin CAAT box alone was unable to stimulate gene expression directed by the LCR and high levels of transcription were obtained only upon inclusion of more upstream beta-globin promoter sequences. In contrast, a construct containing only the A gamma-globin CAAT box region did give high expression levels in K562 cells. Thus, there is a fundamental difference in the way the LCR functions in these two model systems in terms of its requirements at the promoter level.
Delvoye
N L
NL
Institut du Cancer de Montréal, Quebec, Canada.
Destroismaisons
N M
NM
Wall
L A
LA
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D005914:Globins / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers / Q000502:physiology
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D014157:Transcription Factors; D005914:Globins