DNA-dependent adenosine triphosphatase (helicaselike transcription factor) activates β-globin transcription in K562 cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 348-356 ◽  
Author(s):  
Milind C. Mahajan ◽  
Sherman M. Weissman
Keyword(s):  
Transcription Factor ◽  
Developmental Regulation ◽  
Globin Gene ◽  
K562 Cells ◽  
Homologous Sequence ◽  
Gene Promoters ◽  
Conserved Sequence ◽  
Evolutionarily Conserved ◽  
Dna Elements ◽  
Globin Promoter

Correct developmental regulation of β-like globin gene expression is achieved by preferential transcription of a gene at a given developmental stage, silencing of other β-like gene promoters, and competition among these promoters for interaction with the locus control region (LCR). Several evolutionarily conserved DNA elements in the promoters of the β-like genes and LCR have been studied in detail, and the role of their binding factors has been investigated. However, the β-globin promoter includes additional evolutionarily conserved sequences of unknown function. The present study examined the properties of a 21-base pair (bp) promoter-conserved sequence (PCS) located at positions −115 to −136 bp relative to the transcription start site of the β-globin gene. A helicaselike transcription factor (HLTF) belonging to the SWI2/SNF2 family of proteins binds to the PCS and a partly homologous sequence in the enhancer region of the LCR hypersensitive site 2 (HS2). Elevation of the level of HLTF in K562 erythroleukemic cells increases β-promoter activity in transient transfection experiments, and mutations in the PCS that remove HLTF-binding regions abolish this effect, suggesting that HLTF is an activator of β-globin transcription. Overexpression of HLTF in K562 cells does not affect the endogenous levels of γ- and ε-globin message, but it markedly activates β-globin transcription. In conclusion, this study reports a transcription factor belonging to the SWI2/SNF2 family, which preferentially activates chromosomal β-globin gene transcription and which has not previously been implicated in globin gene regulation.

Changes in Globin Gene Methylation and Covalent Histone Modifications of Chromatin Associated with the ε-, γ-, and β-Globin Promoters of the Baboon (P. Anubis) during Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1206-1206
Author(s):  
Donald Lavelle ◽  
Kestas Vaitkus ◽  
Maria Hankewych ◽  
Mahipal Singh ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Rna Pol Ii ◽  
Cpg Sites ◽  
Globin Promoter ◽  
Globin Gene Expression ◽  
Covalent Histone Modifications

Abstract The pattern of globin gene expression during development is conserved in all simian primates, but not in prosimians or other species. Therefore knowledge of the mechanisms regulating globin gene expression in animal models such as the baboon (P. anubis) is directly applicable to human. This investigation addressed the role of chromatin structure in developmental regulation of globin gene expression. DNA methylation of the ε- and γ-gene promoters and covalent histone modifications in chromatin associated with the ε- γ- and β-globin gene promoters have been investigated in 40d fetal primitive nucleated yolk sac-derived RBCs, and definitive erythroid precursor cells from fetal liver (40d to 56d), fetal BM (154d to 160d), and BM from phlebotomized adults. The methylation status of 3 CpG sites in the ε-globin promoter and 5 CpG sites in the γ-globin promoter was analyzed by sequencing 10 cloned PCR products of each sample following bisulfite modification. The ε-globin promoter was unmethylated in 40d primitive yolk-sac derived RBCs. Moderate methylation of the ε-globin promoter was observed in 40d fetal liver (33%: 50%) and was increased in fetal liver samples obtained 2 weeks later in gestation (54d: 76.6%, 56d: 79.1%) to levels observed in late term fetal BM ( 154d: 80%, 156d: 96.6%, 160 d: 93.1%) and adult BM (84.1%; n=2). Methylation of the γ-globin promoter was lowest in 40d primitive RBC (0%) and early fetal liver (40d: 3.1%, 54d: 0%, 56d: 7.1%) and was moderately increased in fetal BM (154d: 38.6%, 156d: 20%, 160d: 30%) compared to adult BM ( 67.3%; n=3). Levels of ac-H3, ac-H4, dimethyl H3 lys4 (H3-dimeK4), dimethyl H3 lys79 (H3-meK79), dimethyl H3 lys36 (H3-meK36), and RNA pol II bound to the ε-, γ-, and β-globin promoters were determined by immunoprecipitation of formaldehyde-fixed, sheared chromatin (ChIP) followed by real time PCR. The amount of RNA pol II, ac-H3, and ac-H4 associated with each globin promoter correlated with developmental-specific gene expression and differed from the pattern of H3-meK79 and H3-meK4 associated with these promoters during development. The amount of H3-meK79 and H3-dimeK4 bound to the the ε- and γ-globin promoters in 40d primitive RBC and fetal liver erythroid precursors (54 and 56d) was 5 times greater than to the β-globin promoter, while similar levels of each (< 2 fold difference) were associated with all three promoters in fetal and adult bone marrow cells. In contrast, the highest level of H3-meK36 was associated with developmentally silenced genes. The amount of H3-meK36 bound to the ε promoter was 2–3 fold higher than to the γ and β promoters in fetal liver (54 and 56d). Similar levels (<2 fold difference) of H3-meK36 were associated with the γ and ε promoters in late term fetal and adult BM and were 2–6 fold greater than bound to the β promoter. We conclude that the chromatin cofiguration of the β-globin locus undergoes distinctive changes associated with both gene activation and silencing during development. Changes in the levels of H3-dimeK4 and H3-meK79 may reflect generalized domain opening, while high levels of ac-H3 and ac-H4 are bound to the promoters of activated genes. In contrast, gene silencing is correlated with increased DNA methylation and enrichment of H3-meK36 bound to the promoters. Thus the baboon model offers unique opportunities to study developmental regulation of globin gene expression.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 353-353 ◽  
Author(s):  
Kenneth R Peterson ◽  
Flavia C Costa ◽  
Halyna Fedosyuk ◽  
Renee Neades ◽  
Johana Bravo de los Rios ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Complex ◽  
Globin Gene ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Zinc Metabolism ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract Abstract 353 Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein. In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of β-Globin Promoter by Erythroid Krüppel-Like Factor

1998 ◽  
Vol 18 (1) ◽  
pp. 102-109 ◽  
Author(s):  
Haruhiko Asano ◽  
George Stamatoyannopoulos
Keyword(s):  
Binding Site ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Sequence Difference ◽  
Optimal Position ◽  
Finger Protein ◽  
Primary Determinant ◽  
Globin Promoter ◽  
Globin Gene Expression

ABSTRACT Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the β-globin gene CACCC box and is essential for β-globin gene expression. EKLF does not activate the γ gene, the CACCC sequence of which differs from that of the β gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the β gene by EKLF, the CACCC boxes of β and γ genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the β promoter carrying a γ CACCC box at a level comparable to that at which it activated the wild-type β promoter, whereas EKLF failed to activate a γ promoter carrying the β CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the β promoter carrying the γ CACCC box, or that EKLF activated the mutated β promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the β or γ promoter determined EKLF specificity, the proximal β CACCC box sequence was created at the position of the β promoter (−140) which corresponds to the position of the CACCC box on the γ promoter. Similarly, the β CACCC box was created in the position of the γ promoter (−90) corresponding to the position of the CACCC box in the β promoter. EKLF retained weak activation potential on the β−140CAC promoter, whereas EKLF failed to activate the γ−90βCAC promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the β promoter by EKLF is determined by the overall structure of the β promoter rather than solely by the sequence of the β gene CACCC box.

scholarly journals In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites

2016 ◽  
Vol 2016 ◽  
pp. 1-27 ◽  
Author(s):  
Kristopher J. L. Irizarry ◽  
Randall L. Bryden
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Coat Color ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Color Variation ◽  
Factor Binding ◽  
Conserved Sequence ◽  
Evolutionarily Conserved

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons.

scholarly journals Restoration of the CCAAT Box or Insertion of the CACCC Motif Activate δ-Globin Gene Expression

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 421-427 ◽  
Author(s):  
Delia C. Tang ◽  
David Ebb ◽  
Ross C. Hardison ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Globin Gene ◽  
Treatment Strategies ◽  
K562 Cells ◽  
Promoter Sequence ◽  
Flanking Sequence ◽  
Globin Promoter ◽  
Ccaat Box ◽  
Globin Gene Expression

Abstract Hemoglobin A2 (HbA2 ), which contains δ-globin as its non–α-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the δ-globin gene in adult erythroid cells, we first compared promoter sequences and found that the δ-globin gene differs from the β-globin gene in the absence of an erythroid Krüppel-like factor (EKLF ) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human δ-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5′ flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the δ promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P &lt; .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type δ promoter. A set of substitutions that create an EKLF binding site centered at −85 bp increased the expression by 26.8-fold and 6.5-fold (P &lt; .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase δ-globin gene expression, with potential future clinical benefit.

Ferritin Heavy Chain, a Repressor of the Human β-Globin Gene That Binds a Conserved Cagtgc Motif, Is Bound to the Repressed β-Globin Promoter In Vivo in K562 Cells and Specifically Binds an Analogous Site in the Mouse βMajor-Globin Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1223-1223
Author(s):  
Robert H. Broyles ◽  
Visar Belegu ◽  
Charles A. Stewart ◽  
Quentin N. Pye ◽  
Austin C. Roth ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Chromatin Immunoprecipitation ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Ferritin Heavy Chain ◽  
Polyclonal Antisera ◽  
Globin Promoter ◽  
F1 Generation

Abstract Ferritin heavy chain (FH), an embryonically-expressed protein in the erythroid lineage, localizes to the nucleus and represses the human adult β-globin promoter in transient expression assays (Broyles et al., PNAS98: 9145, 2001). Recently, we have performed chromatin immunoprecipitation (ChIP) assays with cross-linked chromatin of K562 cells in which the β-globin gene is repressed, using anti-FH polyclonal antisera. These results strongly indicate that FH occupies the repression site (a CAGTGC motif) in vivo. Binding to this -150 site has been previously demonstrated to be required for β-promoter repression in co-transfections. EMSA assays (competitive gel shifts) have revealed that the mouse βMajor-globin promoter has an analogous CAGTGN motif at -160 bp from the cap site that competes specifically with the human CAGTGC site for FH binding. The mouse βMinor-globin promoter lacks the -150/-160 CAGTGN motif and, therefore, the FH binding site. Thus, a human FH transgenic mouse, in which the FH gene is driven by a truncated β-promoter lacking the CAGTGN motif, should express human FH in definitive erythroid cells where the FH would be predicted to repress βMajor-globin but not βMinor-globin. Such a mouse would be predicted to survive but be born with a mild β-thalassemia due to the decreased βMajor/βMinor ratio in its definitive erythroid cells. Preliminary results from the litters of F1 generation FH-tg mice indicate that such is indeed the case, i.e., that human FH functions as a βMajor-globin repressor in vivo.

Differential Modulation of the β-Like Globin Genes by KLFs Isolated with a γ-Globin CACCC Bait.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3637-3637
Author(s):  
Paolo Moi ◽  
Loredana Porcu ◽  
Maria G. Marini ◽  
Isadora Asunis ◽  
Maria G. Loi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Globin Gene ◽  
K562 Cells ◽  
Tissue Expression ◽  
Pcr Analysis ◽  
Globin Genes ◽  
Band Shift ◽  
Hemoglobin Switching ◽  
Specific Complex ◽  
Globin Promoter

Abstract The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger to the β- and ε- than to the γ-CACCC box. On the other hand, KLF11 and 16 did not produce any specific complex in band shift assays with anyone of the globin CACCC boxes. More relevant differences were observed among the factors in the transactivation of single and dual luciferase reporters in both K562 and MEL cells. In these assays, most factors presented peculiar modulatory properties and specific promoter tropism. Several factors presented bidirectional activity displaying in the same time the capacity to stimulate and repress different globin promoters. KLF1 and 4 were the strongest stimulators of the β-globin promoter in both cell lines, whereas KLF2 activated the β-promoter only in K562 cells. KLF1 and especially KLF4 consistently repressed ε-globin expression especially in MEL cells. KLF3 behaved always as a general globin repressor in MEL cells, but acted as a weak stimulator of the γ- and ε-promoter in K562 cells. KLF4 was the strongest inhibitor of the ε-globin gene. KLF13 significantly stimulated the γ-promoter in both cell lines, whereas KLF3, 4 and 6 showed statistically significant stimulation only in MEL cells. By RT-PCR analysis we found that KLFs were highly variable in their tissue expression and that KLF1, 3 and 13 had the highest expression in erythroid tissues. Thus the level of tissue expression should ultimately determine which factors are really active in physiological conditions. Taken together our binding and expression studies suggest that several KLFs have the potential to modulate the activity of the globin genes and that the resulting globin expression will depend on the vectorial sum of the relative activities of the factors expressed at any given time of development. Furthermore, as some KLFs, like KLF1 and 4, exert opposite effects on fetal and adult globin genes, their role in hemoglobin switching may be direct and not only dependent on their ability to mediate promoter competition for the LCR.

Role of gene order in developmental control of human gamma- and beta-globin gene expression

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes

1993 ◽  
Vol 13 (4) ◽  
pp. 2298-2308
Author(s):  
Q Zhang ◽  
P M Reddy ◽  
C Y Yu ◽  
C Bastiani ◽  
D Higgs ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Site Directed Mutagenesis ◽  
Erythroid Cell ◽  
Nuclear Factor ◽  
Sequence Motifs ◽  
Alpha Globin ◽  
Globin Promoter

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1212-1212
Author(s):  
Deepa Manwani ◽  
Mariann Galdass ◽  
James J. Bieker
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Zinc Finger ◽  
Embryoid Body ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Beta Globin Gene ◽  
Body Development ◽  
Globin Promoter

Abstract The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.

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