The beta-globin stage selector element factor is erythroid-specific promoter/enhancer binding protein NF-E4.

Abstract

The analysis of transcriptional regulatory proteins is often hampered because such factors are present in cells in only sparing abundance. Although direct biochemical purification has been successfully applied to the analysis of many of these factors, such methods are labor intensive and expensive. We have developed an alternative strategy to identify and characterize such trans-acting factors and have used it to analyze the proteins that interact with the chicken adult p-globin gene enhancer and promoter. The methodology involves (1) a sensitive 'reverse' radioimmunoassay used for the identification of antibodies to sequence-specific DNA-binding proteins, and (2) a monoclonal antibody-based DNase I footprint selection technique, which unambiguously identifies proteins responsible for particular footprints. Because this methodology relies on the isolation of antibodies to sequence-specific DNA-binding proteins, it should be of general utility in studying any trflns-acting regulatory factor for which a specific DNA-binding sequence can be identified. In the present analysis, we report the identification of a 65-kD protein that is present only in mature definitive (adult) chicken erythroid cells. We show that this protein (termed NF-E4) binds to closely related sequences present in both the p-globin promoter and enhancer. Biochemical analysis of extracts prepared from both nonerythroid and a variety of erythroid cell types suggests that NF-E4 is the trans-acting factor that confers definitive erythrocyte stage-specific transcriptional activation to the adult p-globin gene.

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

“…The final level of regulation, determination of which globin gene isotype is to be expressed at a particular developmental stage [referred to as hemoglobin switching), is elicited by cooperative cis-regulatory interactions between distal control elements (enhancers or DCR/LARI and sequences within physically linked genes Enver et al 1990). In at least one case, this regulation may be due to the binding, within the chicken adult ~-globin gene promoter and enhancer, of a unique developmental stageand tissue-specific erythroid transcription factor, NF-E4 (Gallarda et al 1989).…”

“…Within the chicken ~-globin gene promoter and enhancer, multiple constitutive and tissue-or stage-specific trans-acting factor binding sites have been defined, and their activity has been correlated with function both in vitro and in vivo (Emerson et al 1987(Emerson et al , 1989Choi and Engel 1988;Lewis et al 1988;Nickol and Felsenfeld 1988;Reitman and Felsenfeld 1988;Gallarda et al 1989). The first of the erythrocyte-specific trans-acting factor binding sites to be identified is a prevalent motif, WGATAR (W --T or A; R = G or A), found in common within the human ~/-globin promoter and in the human and chicken ~-globin gene enhancers (Evans et al 1988;Wall et al 1988;Catala et al 1989;Martin et al 1989;Perkins et al 1989).…”

“…Molecular genetic analysis has implicated the presence of several levels of transcriptional control: The first appears to be the ability to form an active chromatin domain [Weintraub and Groudine 1976) to allow the [3-globin gene cluster to be transcribed; such activity may be a reflection of the genetic effect of dominant control regions {DCRsl or locus activation regions (LARs) within the [3-globin gene cluster (Forrester et al 1987; Grosveld et al 1987;Trudel et al 19871. Another level of control appears to be elicited by the tissue-specific transcriptional activity of the [3-globin gene enhancer [Choi and Engel 1986;Hesse et al 1986;Behringer et al 1987; Kollias et al 1987), a complex module whose activity is regulated by the binding of both ubiquitous and tissuespecific factors [Emerson et al 1987; Wall et al 1988;Gallarda et al 1989). The final level of regulation, determination of which globin gene isotype is to be expressed at a particular developmental stage [referred to as hemoglobin switching), is elicited by cooperative cis-regulatory interactions between distal control elements (enhancers or DCR/LARI and sequences within physically linked genes Enver et al 1990).…”

“…Furthermore, cis-regulatory domains appear to exert their effect by binding various combinations of both constitutive and tissue-specific factors to form functional modules; however, only rarely does the effect of deletion of a single protein binding site within a module abrogate the overall physiological effect of the entire regulatory module (e.g., Gallarda et al 1989).…”

Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.

“…For these studies, the parental GATA and GATA/ER cDNAs were inserted into a eukaryotic expression plasmid TFAneo (Federspiel et al 1989;Yamamoto et al 1990), and the activator and reporter plasmids were cotransfected into QT6 quail fibroblasts. To monitor GATA activity, the C3BGH reporter plasmid, which has three copies of the GATA inverted repeat motif of the chicken B-globin enhancer (Emerson et al 1987;Gallarda et al 1989) cloned 5' to the rabbit B-globin TATA box, was used. This artificial promoter directs transcription of the human growth hormone (hGH) gene in the C3[~GH reporter plasmid (Yamamoto et al 1990).…”