scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318 ◽  
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

Functional Analysis of the GATA2 Promoter Shows That Mutations of GATA2 Impair Its Own Transcriptional Regulation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1233-1233 ◽  
Author(s):  
Xabier Cortes-Lavaud ◽  
Miren Maicas ◽  
Iria Vazquez ◽  
Carmen Vicente ◽  
Leire Urquiza ◽  
...  
Keyword(s):  
Binding Sites ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Regulatory Elements ◽  
Full Length ◽  
Recurrent Event ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoter Construct

Abstract Abstract 1233 GATA2 encodes a transcription factor with essential functions in hematopoiesis. Somatic mutations of GATA2 have been reported in patients with chronic myeloid leukemia (CML) with blast crisis, and in bi-CEBPA-positive acute myeloid leukemia (AML); moreover, our group and others have shown that overexpression of this gene is a recurrent event associated with poor prognosis in AML. Several recent studies report mutations in the GATA2 gene in three different familial syndromes characterized by predisposition to myelodysplastic syndrome (MDS) and AML. Despite some differences, these mutations are very similar, and in some cases identical. This implies that individual mutations, although located in similar regions, may differentially affect GATA2 function. Therefore, additional research is required to explain why similar mutations lead to different syndromes (Hyde and Liu, 2011). On the other hand, it has been extensively studied in murine models that GATA2 activates its own transcription by binding to regions located at −2.8 and −1.8 kb from the transcription start site (TSS). We hypothesized that these reported GATA2 mutations could alter the GATA2 autoregulatory loop, affecting the transcription of GATA2. With this aim, we first aligned the murine and human GATA2 promoters in search for homologous GATA2 binding sites. Regions containing the cis-regulatory elements located at −2.8 and −1.8 kb from IS exon TSS in the murine promoter were highly homologous to two regions in the human promoter, with two putative GATA2 binding sites located at −3.4 and −2.4 kb from IS TSS, respectively. ChIP-qPCR assays showed that GATA2 binds to these sites in the human GATA2 promoter. To assess the ability of both wild-type and GATA2 mutations to regulate its own transcription, we transfected these GATA2 gene variants along with different GATA2 promoter constructs into HEK293T cells, and performed luciferase reporter assays. Wild-type GATA2 activated its transcription through the −2.4 kb site; however, it was not able to activate the full length promoter construct containing both the −3.4 and −2.4 sites. CEBPA binding sites near the −3.4 site could explain these results, since it has been reported that expression of GATA2 is transcriptionally repressed by CEBPA in a DNA binding-dependent manner. The T354M mutant activated GATA2 transcription in a similar manner than the GATA2 wild-type, raising the question about the complex function of T354M. On the contrary, del355T was totally incapable of sustain any activation of GATA2. Finally, the L359V mutation, present in 10% of CML cases with blast crisis, was able to activate the GATA2 promoter, even the full length promoter construct that contains both −3.4 and −2.4 sites, supporting that L359V is a gain-of-function mutation. In summary, GATA2 mutations had different effects on the GATA2 promoter that could affect the dose of GATA2. Expression of GATA2 is critical at various stages of hematopoiesis and since it in part determines the fate of distinct myeloid lineages, this could alter normal hematopoiesis. Moreover, as happened with GATA2, mutant GATA2 proteins could affect the expression of other targets of GATA2, as SCL, BMP4, PU.1, WT1 and others. Studies to further clarify these questions are in progress. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4217-4222 ◽  
Author(s):  
Rakesh Singal ◽  
Jane M. vanWert ◽  
Larry Ferdinand
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Histone H4 ◽  
Luciferase Reporter Gene ◽  
Cpg Dinucleotides ◽  
Expression Construct ◽  
Globin Promoter

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonicαπ-globin promoter was unmethylated, andαπ-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the απ promoter associated with loss of expression of απ-globin gene was seen during development in primary erythroid cells. A 315-bpαπ-globin promoter region was cloned in an expression construct (απpGL3E) containing a luciferase reporter gene and SV40 enhancer. The απpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of απpGL3E plasmid andαπ-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bpαπ-globin gene promoter fragment formed amethyl cytosine-binding proteincomplex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with theαπ-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

scholarly journals Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 990-999 ◽  
Author(s):  
MJ Ulrich ◽  
TJ Ley
Keyword(s):  
Transcription Initiation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Cis Acting ◽  
Promoter Function ◽  
Gamma Globin ◽  
Globin Promoter

Abstract We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0254466
Author(s):  
Ting-Yun Chen ◽  
Xiaoyun Li ◽  
Gillian C. Goobie ◽  
Ching-Hsia Hung ◽  
Tin-Kan Hung ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Sites ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Reporter System ◽  
Reduced Activity

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin’s antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

Regulation of DHP receptor expression by elements in the 5′-flanking sequence

2000 ◽  
Vol 278 (4) ◽  
pp. H1153-H1162 ◽  
Author(s):  
Lei Liu ◽  
Q. Ivy Fan ◽  
Mohamad R. El-Zaru ◽  
Kathleen Vanderpool ◽  
Ronald N. Hines ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Receptor Expression ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Subunit Gene ◽  
Luciferase Reporter Gene ◽  
Response Element ◽  
Flanking Region

The α1-subunit of the cardiac/vascular Ca2+channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac α1C-subunit gene, we have cloned a 2.3-kb fragment containing the 5′-flanking sequences and identified the α1C-subunit gene transcription start site. The rat α1C-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of α1C-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5′-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5′-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca2+ channel α1C-subunit gene. Electrophoretic mobility shift assays identified a region of the α1C-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

Acetylation of an Ets Transcription Factor PU.1 Suppresses Its Transcriptional Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2402-2402
Author(s):  
Fumihiko Mouri ◽  
Junichi Tsukada ◽  
Akiyoshi Fukamizu ◽  
Yoshiya Tanaka
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activity ◽  
Hematopoietic Cells ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Wild Type ◽  
Il1b Gene

Abstract PU.1, a member of the Ets family transcription factors, is expressed restrictively in hematopoietic cells including monocytes and macrophages, and plays critical roles in the inflammatory responses and the development of hematopoietic cells. CREB-binding protein (CBP) regulates transcription by acetylating not only histones but also certain transcription factors. Here, we demonstrated that a specific inhibitor of histone deacetylases, trichostatin A (TSA) inhibits PU.1 transcriptional activity in monocytes and further showed that deletion of a histone acetyltransferase (HAT) domain of CBP resulted in synergistic cooperativity between CBP and PU.1. When human monocytic cells THP-1 were treated with TSA, our immunoprecipitaion and western blot assay showed that TSA enhanced PU.1 acetylation. Next, we investigated the effect of TSA on the transcriptional regulation of PU.1-dependent gene promoters such as the human prointerleukin 1β (IL1B) gene and the human granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene in transient transfection studies. Two distinct luciferase reporter plasmids (Luc) for the IL1B gene promoter and the GM-CSFRα gene promoter, IL1B-Luc and GM-CSFRα-Luc were used. When these plasmids were transiently transfected into THP-1 cells, TSA suppressed LPS-induced activities for the IL1B promoter and the GM-CSFRα promoter in a dose-dependent manner. In contrast, when NF-κB luciferase reporter, NF-κB-Luc was transfected into THP-1 cells, TSA synergistically increased LPS-induced NF-κB activities. Moreover, when a PU.1 expression vector, pECEPU.1 was cotransfected into PU.1-deficient murine thymocytes EL4 along with either IL1B-Luc or GM-CSFRα-Luc. The PU.1-induced promoter activities were strongly suppressed through TSA treatment. FACS analysis further indicated that TSA suppressed LPS-induced expression of IL-1β and GM-CSFRα proteins. In addition, our EMSA data showed that TSA treatment did not affect DNA binding activity of PU.1 to the IL1B promoter. PU.1 has been shown to interact physically with CBP to transactivate their target genes. In our study, expression vectors for CBP wild-type or with a deletion of its HAT domain was cotransfected into EL4 cells along with IL1B-Luc and pECEPU.1. The HAT activity-deficient mutant showed synergistic transcriptional activity with PU.1 more strongly than the wild-type CBP. In this regard, our GST-pulldown assay showed that deletion of CBP HAT domain did not change binding affinity of CBP for PU.1. Our results propose a novel molecular mechanism by which PU.1-dependent genes is negatively regulated by HAT-induced acetylation in monocytes.

scholarly journals Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 990-999 ◽  
Author(s):  
MJ Ulrich ◽  
TJ Ley
Keyword(s):  
Transcription Initiation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Cis Acting ◽  
Promoter Function ◽  
Gamma Globin ◽  
Globin Promoter

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

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