The rat βbminy-globin promoter: nuclear protein factors and erythroid-specific induction of transcriptionRID="†"ID="†" Research Article (original) (raw)
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The EMBO journal, 1988
We have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF-E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT-box factor (CP1). Deletion experiments suggest that only the combination of NF-E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta-globin promoter in mouse erythroleukaemia (MEL) cells.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human /3-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Spl and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human /3-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the/3-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can cooperatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from-103 in the human /3-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human /3-globin cluster.
Proceedings of the National Academy of Sciences, 2000
The programmed expression of the five -like globin genes (, A ␥, G ␥, ␦, and ) is characterized by a series of switches that are developmentally regulated. The A ␥and G ␥-(fetus) to -globin (adult) switch depends on transcription factor erythroid Krü ppellike factor (EKLF), which, like Sp1, binds to CACCC boxes. EKLF is essential for the expression of the -globin but not the ␥-globin gene. Because both ␥-globin and -globin promoters contain the CACCC box, and their promoter elements are similar, it is not known why the two promoters behave so differently. In this report, we searched for the functional differences between the two promoters by studying their ability to recruit EKLF. We used the in vivo PIN*POINT assay to show that EKLF is recruited to the -globin promoter but not to the ␥-globin promoter. We show that this selectivity is a result of differences in surrounding promoter elements and not CACCC box alone. One of the differences between the two promoters with a functional consequence is the CCTTG repeat that is present in the ␥-globin promoter but not in the -globin promoter. The repeat, when inserted in the -globin promoter, decreases EKLF recruitment to and activity of the -globin promoter, suggesting that the repeat functions as a suppressor element. The CCTTG repeat can also suppress the SV40 promoter in cis, and the suppressor factor binding to the repeat can be squelched with a plasmid containing a high copy number of the repeat. These findings may have implications in designing drug targets for treatment of -globin disorders.
Characterization of the human β‐globin downstream promoter region
Nucleic Acids Research, 2003
The human b-globin gene is abundantly expressed speci®cally in adult erythroid cells. Stage-speci®c transcription is regulated principally by promoter proximal cis-regulatory elements. The basal promoter contains a non-canonical TATA-like motif as well as an initiator element. These two elements have been shown to interact with the TFII-D complex. Here we show that in addition to the TATA and initiator elements, conserved E-box motifs are located in the b-globin downstream promoter. One of the E-box motifs overlaps the initiator and this composite element interacts with USF1 and TFII-I in vitro. Another E-box, located 60 bp 3¢ to the transcription initiation site, interacts with USF1 and USF2. Mutations of either the initiator or the downstream E-box impair transcription of the b-globin gene in vitro. Mutations of a putative NF-E2-binding site in the downstream promoter region do not affect transcription in vitro. USF1, USF2, TFII-I and p45 can be crosslinked to a b-globin promoter fragment in MEL cells in vivo, whereas only TFII-I and USF2 crosslink to the b-globin gene in K562 cells. The summary data demonstrate that in addition to the well-characterized interactions of the TFII-D complex with the basal promoter, E-box motifs contribute to the ef®cient formation of transcription complexes on the adult b-globin gene.
Molecular and cellular biology, 1995
Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the -globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer--globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.
Characterization of the human beta-globin downstream promoter region
Nucleic Acids Research, 2003
The human b-globin gene is abundantly expressed speci®cally in adult erythroid cells. Stage-speci®c transcription is regulated principally by promoter proximal cis-regulatory elements. The basal promoter contains a non-canonical TATA-like motif as well as an initiator element. These two elements have been shown to interact with the TFII-D complex. Here we show that in addition to the TATA and initiator elements, conserved E-box motifs are located in the b-globin downstream promoter. One of the E-box motifs overlaps the initiator and this composite element interacts with USF1 and TFII-I in vitro. Another E-box, located 60 bp 3¢ to the transcription initiation site, interacts with USF1 and USF2. Mutations of either the initiator or the downstream E-box impair transcription of the b-globin gene in vitro. Mutations of a putative NF-E2-binding site in the downstream promoter region do not affect transcription in vitro. USF1, USF2, TFII-I and p45 can be crosslinked to a b-globin promoter fragment in MEL cells in vivo, whereas only TFII-I and USF2 crosslink to the b-globin gene in K562 cells. The summary data demonstrate that in addition to the well-characterized interactions of the TFII-D complex with the basal promoter, E-box motifs contribute to the ef®cient formation of transcription complexes on the adult b-globin gene.
Genes & Development, 2006
We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine -globin locus during erythroid differentiation. Murine fetal liver cells were separated into distinct erythroid maturation stages by fluorescence-activated cell sorting, and the nuclear position of the locus was determined at each stage. We find that the -globin locus progressively moves away from the nuclear periphery with increasing maturation. Contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells,  major -globin expression begins at the nuclear periphery prior to relocalization. However, relocation of the locus to the nuclear interior with maturation is accompanied by an increase in  major -globin transcription. The distribution of nuclear polymerase II (Pol II) foci also changes with erythroid differentiation: Transcription factories decrease in number and contract toward the nuclear interior. Moreover, both efficient relocalization of the -globin locus from the periphery and its association with hyperphosphorylated Pol II transcription factories require the locus control region (LCR). These results suggest that the LCR-dependent association of the -globin locus with transcriptionally engaged Pol II foci provides the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.
Promoter sequences required for function of the human gamma globin gene in erythroid cells
The EMBO journal, 1986
The human gamma- and beta-globin genes are expressed during the fetal and adult developmental periods, respectively. Differences in the sequences of their promoters may be relevant to their developmental regulation. The gamma globin gene promoter was found to be stronger than that of the beta gene. In HeLa cells co-transfected with plasmids containing the two genes, transcripts arising from the gamma promoter accumulated to a level 3-fold higher than those initiated from the beta-promoter. We have recently shown that deletion of the most distal of the conserved elements of the promoter (CACCC) reduces function to 25% of the wild-type, whereas the removal of the proximal of the two duplicated (CCAAT) elements increases promoter function 2- to 4-fold in HeLa cells during transient gene expression. Both the wild-type promoter and the truncation and linker-scanning mutants from which one of the two duplicated "CCAAT' elements had been removed, exhibited regulated expression whe...
Beta-Globin Dominant Control Region Interacts Differently with Distal and Proximal Promoter Elements
Genes & Development, 1990
We have studied the interaction between the dominant control region (DCR) and the promoter of the human []-globin gene. Expression analysis in MEL cells has revealed that the DCR contains a number of elements capable of replacing the upstream (-250 to-100) erythroid-specific region of the promoter. The DCR strongly stimulates expression from a promoter possessing only a TATA box. However, this basic level of transcription is not induced upon erythroid differentiation of the cells. Mutational analysis of the minimal (-100, noninducible) promoter shows that only the combination of the DCR and the CAC/CCAAT elements provides erythroid-specific transcription. These regions act synergistically to produce full regulated expression during erythroid differentiation.
DNA sequences required for regulated expression of β-globin genes in murine erythroleukemia cells
Cell, 1984
We introduced into MEL cells rabbit /3-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human @-globin gene and noninducible human y-globin gene or the murine H-2K"'"' class I MHC gene. Sl nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit @-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human B-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of they or H2Kbm' genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5'7-3'8 and 5'H2-3'8 hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate 8-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site.