scholarly journals Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

1988 ◽  
Vol 8 (8) ◽  
pp. 3215-3226 ◽  
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
S S Banerji ◽  
M Sheffery
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Globin Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Murine Erythroleukemia

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene

1988 ◽  
Vol 8 (8) ◽  
pp. 3215-3226
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
S S Banerji ◽  
M Sheffery
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Globin Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Murine Erythroleukemia

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

scholarly journals Transcriptional promiscuity of the human alpha-globin gene.

1989 ◽  
Vol 9 (1) ◽  
pp. 241-251 ◽  
Author(s):  
E Whitelaw ◽  
P Hogben ◽  
O Hanscombe ◽  
N J Proudfoot
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Multiple Copies ◽  
Positive Effect

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

scholarly journals Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

1989 ◽  
Vol 9 (6) ◽  
pp. 2606-2614 ◽  
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
M Sheffery
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Sequence Motif ◽  
Globin Genes ◽  
Beta Globin ◽  
Alpha Globin ◽  
Murine Erythroleukemia

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.

scholarly journals Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro.

1990 ◽  
Vol 10 (11) ◽  
pp. 5958-5966 ◽  
Author(s):  
C G Kim ◽  
S L Swendeman ◽  
K M Barnhart ◽  
M Sheffery
Keyword(s):  
Dna Sequence ◽  
Gene Transcription ◽  
Binding Sites ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Nuclear Extracts ◽  
Alpha Globin ◽  
Transcription In Vitro ◽  
Murine Erythroleukemia

We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes

1989 ◽  
Vol 9 (6) ◽  
pp. 2606-2614
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
M Sheffery
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Sequence Motif ◽  
Globin Genes ◽  
Beta Globin ◽  
Alpha Globin ◽  
Murine Erythroleukemia

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.

Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro

1990 ◽  
Vol 10 (11) ◽  
pp. 5958-5966
Author(s):  
C G Kim ◽  
S L Swendeman ◽  
K M Barnhart ◽  
M Sheffery
Keyword(s):  
Dna Sequence ◽  
Gene Transcription ◽  
Binding Sites ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Nuclear Extracts ◽  
Alpha Globin ◽  
Transcription In Vitro ◽  
Murine Erythroleukemia

We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.

Transcriptional promiscuity of the human alpha-globin gene

1989 ◽  
Vol 9 (1) ◽  
pp. 241-251
Author(s):  
E Whitelaw ◽  
P Hogben ◽  
O Hanscombe ◽  
N J Proudfoot
Keyword(s):  
Cell Lines ◽  
Transcriptional Activity ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Multiple Copies ◽  
Positive Effect

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication

1992 ◽  
Vol 12 (6) ◽  
pp. 2514-2524 ◽  
Author(s):  
Z S Guo ◽  
M L DePamphilis
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Replication ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Activation Domains ◽  
Auxiliary Component ◽  
Cell Extracts

The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

scholarly journals Human embryonic zeta-globin gene expression in mouse-human hybrid erythroid cell lines

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1212-1217 ◽  
Author(s):  
HY Luo ◽  
AB Deisseroth ◽  
DH Chui
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Hybrid Cells ◽  
Globin Chains ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Zeta Globin Gene ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

Abstract The human alpha-globin-like embryonic zeta-globin chains are present in abundance during the first 5 to 6 weeks of gestation. Subsequently, zeta-globin chains are present in fetal blood at a very low level, which is supplanted by the expression of alpha-globin chains. Adult individuals who are carriers of the (--SEA/) alpha-thalassemia deletion, in contrast to normal adults, have low levels of embryonic zeta-globin chains in their circulating erythrocytes. In this investigation, we constructed stable mouse-human hybrid cells with murine erythroleukemia cells bearing human chromosome 16, with either the normal alpha-globin gene cluster (alpha alpha/) or the (--SEA/) type of alpha-thalassemia deletion. The results on the human zeta- globin gene expression in these hybrid cells indicate that murine adult erythroid transcription factors can induce the expression of human embryonic zeta-globin gene is cis to the (--SEA/) deletion, in parallel with the endogenous mouse alpha-globin gene expression. These data also show the importance of the DNA sequences within the (--SEA) deletion in regulating the expression of zeta-globin gene in cis during normal human hemoglobin ontogeny.

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