Context-Specific KLF-1 Mediated Transcriptional Activation at the Alpha Hemoglobin-Stabilizing Protein and Dematin Promoters in Erythroid Cells.

Abstract Abstract 461 Krüppel-like factor 1 (KLF1) is essential for erythroid gene expression. Key molecular mechanisms modulated by this transacting factor have been elucidated at the b-globin locus. KLF1 has been associated with recruitment of SWI/SNF and RNA polymerase (PolII) complexes necessary for chromatin remodeling and gene transcription respectively, and for facilitating the apposition of the promoter with the far-upstream locus control region. More recently, KLF1 has been implicated in the regulation of an erythroid-specific gene program unlinked to the b-globin locus. Coordinated expression of these genes, including Alpha Hemoglobin-Stabilizing Protein (AHSP), a factor required for globin tetramer stability, and the red cell membrane protein Dematin, are critical for erythroid ontogeny. To compare the role(s) of KLF1 at these loci, we have used a unique 4-OH-Tamoxifen (4-OHT) inducible erythroid cell line, which facilitates the characterization of the temporal kinetics of KLF1-dependent erythroid gene activation. In preliminary experiments, we observed that KLF1 binding was maximal at the three loci within 60 minutes of 4-OHT induction. AHSP and dematin primary RNA transcripts followed similar kinetics, being maximal at 60-90 minutes post-induction. In contrast, b-globin gene transcription reached a plateau 4-6 hours post-induction. From these observations, we hypothesized that transcriptional activation at AHSP and dematin differs from that observed at the b-globin cluster. Consistent with this hypothesis, we observed significant differences in chromatin remodeling at the three loci. At the b-globin promoter, we observed a small but statistically significant increase in DNaseI sensitivity, a measure of chromatin remodeling, with KLF1 binding. In contrast, we observed a complete loss of DNaseI resistance after KLF1 binding at the AHSP and dematin promoters. Consistent with these findings, we observed a five-fold reduction in histone H3 occupancy at the AHSP and dematin promoters, contrasting with no significant change in occupancy at the b-promoter. Importantly, these differences were not observed in regions 1-5 kb upstream of the promoters. These observations, coupled with similar differences in DNaseI hypersensitivity and histone occupancy in fetal liver erythroblasts from wild type and KLF1-null mice, suggest a profound difference in the mechanisms of chromatin remodeling at KLF1-dependent erythroid gene loci. To explore the potential mechanisms underlying these differences in chromatin accessibility, we examined the kinetics of recruitment of other transacting factors and co-activators to the three loci. We observed similar increases in binding of serine-5 phosphorylated PolII, GATA-1, and p45NF-E2 at the promoters. In contrast, binding of BRG1, the core ATPase component of the SWI/SNF complex differed between the b–promoter and the other erythroid genes. Although BRG1 binding was co-incident with KLF1 binding to the b-gene, we observed significant albeit weak binding of this complex to the AHSP and Dematin promoters only after maximal gene transcription had occurred. Our results suggest that different KLF1 multiprotein complexes are recruited to remodel target gene promoters in vivo. Furthermore, we propose that KLF1's chromatin remodeling capabilities are not limited to the recruitment of the SWI/SNF complexes Disclosures: No relevant conflicts of interest to declare.

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Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.

Blood ◽

10.1182/blood.v108.11.365.365 ◽

2006 ◽

Vol 108 (11) ◽

pp. 365-365 ◽

Cited By ~ 1

Author(s):

Valerie M. Jansen ◽

Shaji Ramachandran ◽

Aurelie Desgardin ◽

Jin He ◽

Vishwas Parekh ◽

...

Keyword(s):

Gene Expression ◽

Gene Transcription ◽

Globin Gene ◽

Erythroid Cell ◽

Gene Promoters ◽

Globin Genes ◽

Context Specific ◽

High Level ◽

Kinetics Of ◽

Globin Gene Expression

Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.

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Distinct Domains of Erythroid Krüppel-Like Factor Modulate Chromatin Remodeling and Transactivation at the Endogenous β-Globin Gene Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.161-170.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 161-170 ◽

Cited By ~ 43

Author(s):

R. Clark Brown ◽

Scott Pattison ◽

Janine van Ree ◽

Elise Coghill ◽

Andrew Perkins ◽

...

Keyword(s):

Chromatin Remodeling ◽

Gene Transcription ◽

Transcriptional Activation ◽

Globin Gene ◽

Regulatory Elements ◽

Binding Studies ◽

Amino Terminal ◽

Cellular Models

ABSTRACT Characterization of the mechanism(s) of action of trans-acting factors in higher eukaryotes requires the establishment of cellular models that test their function at endogenous target gene regulatory elements. Erythroid Krüppel-like factor (EKLF) is essential for β-globin gene transcription. To elucidate the in vivo determinants leading to transcription of the adult β-globin gene, functional domains of EKLF were examined in the context of chromatin remodeling and transcriptional activation at the endogenous locus. Human EKLF (hEKLF) sequences, linked to an estrogen-responsive domain, were studied with an erythroblast cell line lacking endogenous EKLF expression (J2eΔeklf). J2eΔeklf cells transduced with hEKLF demonstrated a dose-dependent rescue of β-globin transcription in the presence of inducing ligand. Further analysis using a series of amino-terminal truncation mutants of hEKLF identified a distinct internal domain, which is sufficient for transactivation. Interestingly, studies of the chromatin structure of the β-promoter revealed that a smaller carboxy-terminal domain generated an open promoter configuration. In vitro and in vivo binding studies demonstrated that this region interacted with BRG1, a component of the SWI/SNF chromatin remodeling complex. However, further study revealed that BRG1 interacted with an even smaller domain of EKLF, suggesting that additional protein interactions are required for chromatin remodeling at the endogenous β-promoter. Taken together, our findings support a stepwise process of chromatin remodeling and coactivator recruitment to the β-globin promoter in vivo. The J2eΔeklf inducible hEKLF system will be a valuable tool for further characterizing the temporal series of events required for endogenous β-globin gene transcription.

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Hipertrofia muscular esquelética humana induzida pelo exercício físico/Exercise-induced human skeletal muscle hypertrophy

REVISTA CIÊNCIAS EM SAÚDE ◽

10.21876/rcsfmit.v1i2.40 ◽

2011 ◽

Vol 1 (2) ◽

pp. 52-61

Author(s):

Bernardo Neme Ide ◽

Fernanda Lorenzi Lazarim ◽

Denise Vaz de Macedo

Keyword(s):

Protein Synthesis ◽

Resistance Training ◽

Signaling Pathways ◽

Gene Transcription ◽

Satellite Cells ◽

Physical Training ◽

Molecular Mechanisms ◽

Enzyme Activation ◽

Specific Gene ◽

Adaptation Process

A resposta adaptativa ao treinamento físico é determinada pelo tipo, volume e frequência de aplicação dos estímulos, que ativam vias de sinalização distintas, a transcrição de genes específicos e posterior síntese protéica. O treinamento resistido está relacionado à ativação da enzima mTOR, proporcionada pelo hormônio IGF-1 e estimulada pela insulina, quando um carboidrato é consumido após a atividade física. Estas vias de sinalização levam à inibição da transcrição de genes relacionados à atrofia e aumento da síntese de proteínas contráteis e metabólicas, proporcionando um aumento da massa muscular, conhecido como hipertrofia. Atualmente, evidências sugerem que, além das sinalizações dos hormônios, os estímulos mecânicos (mecanotransdução) também podem influenciar a ativação gênica durante o processo hipertrófico. A ativação de células satélites, proporcionada pelo estresse mecânico, fatores de crescimento, radicais livres e citocinas é de suma importância para o crescimento muscular. Devido à relevância deste assunto, o presente trabalho traz uma revisão da literatura a respeito dos processos envolvidos na resposta hipertrófica, em decorrência do treinamento físico. Embora o processo hipertrófico seja bastante estudado, os mecanismos moleculares, tanto em nível gênico quanto protéico, envolvidos no processo adaptativo ainda não são totalmente compreendidos. Neste sentido, o avanço nas técnicas de biologia molecular como genômica, transcriptoma e proteômica abrem caminhos para futuras investigações nesta área.Palavras-chave: treino resistido, adaptações ao treinamento de força, células satélites, IGF-1, síntese protéica.The adaptation process to physical training is determined by the type, volume and frequency of stimulation, activating distinct signaling pathways, specific gene transcription and then protein synthesis. Resistance-training is related to mTOR enzyme activation induced by IGF-1 and stimulated by insulin when carbohydrates are consumed after physical activity. These pathways, may lead to the inhibition of gene transcription related to atrophy and the increment of contractile and metabolic protein synthesis causing an increase on muscle mass known as hypertrophy. Presently, there is evidence to suggest that besides hormone signaling pathways, mechanical stimulation (mechanotransduction) may also influence the gene activation during the hypertrophic process. The satellite cells activation induced by mechanical stress, growth factors, free radicals, and cytokines is crucial for muscle growth. Due to the importance of this topic, the present study, proposes a literature review about the processes related to the hypertrophic responses to physical training. Despite the frequent studies on the hypertrophic process, the molecular mechanisms (both at gene and protein levels) involved in the adaptation process is yet to be fully understood. Thus, advances in molecular biology techniques such as genomic, transcriptoma and proteomic open ways for future investigations in this area.Key words: Resistance-training, strength training adaptations, satellite cells, IGF-1, protein synthesis.

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Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene

The EMBO Journal ◽

10.1093/emboj/19.21.5875 ◽

2000 ◽

Vol 19 (21) ◽

pp. 5875-5883 ◽

Cited By ~ 22

Author(s):

I. M. Gavin

Keyword(s):

Chromatin Remodeling ◽

Transcriptional Activation ◽

Specific Gene ◽

A Cell

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An Erythoid-Specific Component of the Rhesus Stage Selector Protein, rNF-E4, Modulates γ-Globin Gene Expression Specifically.

Blood ◽

10.1182/blood.v108.11.1599.1599 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1599-1599

Author(s):

Ruiqiong Wu ◽

Aurelie Desgardin ◽

Stephen M. Jane ◽

John M. Cunningham

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Globin Gene ◽

Primate Species ◽

Pcr Analysis ◽

Gene Promoters ◽

Fetal Stage ◽

Globin Gene Expression ◽

Selector Protein

Abstract Understanding the molecular mechanisms that regulate γ-globin gene expression is essential for development of new therapeutic strategies for individuals with sickle cell disease and β-thalassemia. We have previously identified a tissue- and developmentally- specific multiprotein transacting factor complex, the human stage selector protein (SSP), which facilitates the interaction of the g-globin gene promoters with the upstream locus control region enhancer in fetal erythoid cells. This complex interacts with the stage selector element (SSE) in the proximal g-globin promoter, a regulatory motif phylogenetically conserved in primate species with a distinct fetal stage of β-globin like gene expression. Given these observations, we hypothesized that a similar complex modulates γ-globin in the rhesus macaque, a non-human primate model that has been utilized to study β-globin like gene expression. We focused our efforts on NF-E4, given that a human isoform of this factor confers erythroid and fetal specificity to the SSP complex. Fetal liver erythroblasts were obtained from rhesus embryos and analyzed by reverse transcriptase(RT)-PCR analysis for NF-E4 expression. NF-E4 like transcripts were identified in day 60, 80 and 120 embryonic erythroblasts, but not other rhesus tissues, demonstrating an erythroid-specific pattern of expression. Utilizing 5′ RACE, we cloned a full length NF-E4 transcript, identifying an open reading frame encoding a 131 amino acid polypeptide. This 20kD polypeptide shares a high degree of homology with human NF-E4, especially in its carboxy-terminal domain. Like human NF-E4, GST pulldown chromatography confirmed the ability of the rhesus factor to interact directly with CP2 and ALY, the other core components of the SSP. To evaluate rNF-E4 function in vivo, we utilized retrovirally mediated gene transfer to enforce expression of this factor in K562 cells, a model of human fetal erythropoiesis. Initial co-immunoprecipitation studies confirmed the in vivo interaction of rNF-E4 with other components of the SSP. Interestingly, we observed a specific 3-fold induction of γ-globin gene expression in rNF-E4 expressing cells when compared to controls. Moreover, we demonstrated that, like enforced expression of human NF-E4, rNF-E4 induced a significant increase in ε-globin gene expression. Taken together, our results suggest a conservation of NF-E4 expression and function in species with a fetal stage of globin gene expression. Moreover, the identification of rNF-E4 provides a platform for the pre-clinical development of therapeutic agents that induce high levels of NF-E4 in adult erythroblasts.

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Molecular regulation of CC-chemokine receptor 3 expression in human T helper 2 cells

Blood ◽

10.1182/blood.v98.8.2568 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2568-2570 ◽

Cited By ~ 17

Author(s):

Emmanuel Scotet ◽

Susanne Schroeder ◽

Antonio Lanzavecchia

Keyword(s):

T Cells ◽

Chromatin Remodeling ◽

Chemokine Receptor ◽

Molecular Mechanisms ◽

Regulatory Element ◽

Th2 Cells ◽

Specific Gene ◽

Upstream Sequence ◽

T Helper ◽

Cc Chemokine

Abstract In developing T helper 1 (Th1) and Th2 cells the acquisition of effector function is intimately connected with the acquisition of new migratory capacities, as exemplified by differential expression of chemokine receptors. This study investigates the molecular mechanisms responsible for Th2-restricted expression of the CC-chemokine receptor 3 (CCR3). The minimal promoter in T cells was identified in the −149 base pair (bp) upstream sequence that contains a positive regulatory element. A strong negative element was also localized in the flanking intronic sequence. The study further investigates the role of chromatin remodeling in the regulation of this Th2-specific gene. Drugs that affect the chromatin structure facilitate CCR3 expression in T cells. Furthermore, in differentiating Th2 cells, selected regions are associated with acetylated-H3 histones and become more accessible to DNase I. These results suggest that in Th2 cells both cytokine production and migratory capacity are regulated through a similar mechanism involving chromatin remodeling.

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p45NFE2 Is a Negative Regulator of Erythroid Proliferation Which Contributes to the Progression of Friend Virus-Induced Erythroleukemias

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.73-80.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 73-80 ◽

Cited By ~ 18

Author(s):

You-Jun Li ◽

Rachel R. Higgins ◽

Brian J. Pak ◽

Ramesh A. Shivdasani ◽

Paul A. Ney ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Globin Gene ◽

Negative Regulator ◽

Erythroid Cell ◽

Specific Gene ◽

Globin Genes ◽

Friend Virus ◽

Wild Type

ABSTRACT In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific genep45 NFE2 . Frequent disruption ofp45 NFE2 due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV inp45 NFE2 mutant mice. Sincep45 NFE2 homozygous mice mostly die at birth, erythroleukemia was induced in +/− and +/+ mice. We demonstrate that +/− mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/− mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/− and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/− mice. Establishment in culture was associated with the loss of the remaining wild-typep45 NFE2 allele in 9 of 10 of these cell lines. The loss of a functional p45NFE2 in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-typep45 NFE2 in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression ofp45 NFE2 in these cells also slows tumor growth in vivo. These results indicate thatp45 NFE2 functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2298-2308.1993 ◽

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.

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Severe Impairment of γ-Globin Gene Silencing in an Asymptomatic Adult Patient Homozygous for the Codon 8 (–AA) Frame-Shift β0-Thalassemia Mutation

Blood ◽

10.1182/blood.v120.21.1022.1022 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1022-1022

Author(s):

Andrew Campbell ◽

Hong-yuan Luo ◽

Katherine Benson ◽

Lance Davis ◽

Patrick G. Gallagher ◽

...

Keyword(s):

Gene Silencing ◽

Gene Cluster ◽

Gene Transcription ◽

Globin Gene ◽

Minor Allele ◽

Reference Sequence ◽

Compound Heterozygous ◽

Gene Promoters ◽

Globin Gene Cluster ◽

Thalassemia Mutation

Abstract Abstract 1022 Beta-thalassemia is caused by β-globin gene mutations that result in either markedly decreased (β+) or absent (β0) β-globin chain production. Patients who are either homozygous or compound heterozygous for β-thalassemia mutations are severely anemic and require chronic RBC transfusions. Concomitant inheritance of an α -thalassemia mutation or increased HbF (α 2γ2) expression can ameliorate the disease severity. Surprisingly, many patients homozygous for the codon 8 (–AA) β0-thalassemia mutation are mildly anemic with over 95% HbF [Altay & Gürgey, Ann NY Acad Sci 612:81, 1990]. We now report one such patient, a 20-year old man of Iraqi ancestry who was found to have splenomegaly but was otherwise well. His hemoglobin was 12.4 g/dL, MCV 77 fL, reticulocyte count 3.2%. Hemoglobin analysis by HPLC revealed HbF 98%, HbA2 2%. Multiplex ligation-dependent probe amplification (MLPA) of his β-globin gene cluster confirmed that he did not have a large deletion as found in some patients with hereditary persistence of fetal hemoglobin (HPFH), nor γ-globin gene quadruplication. In addition, he was heterozygous for the α 2 IVSI donor splice site 5 bp deletion (–GTGAG), the α Hph thalassemia mutation. To determine the genetic basis for his persistently high HbF expression, his γ-globin gene promoters were sequenced and no HPFH point mutation was found. We next assessed the status of his 3 major HbF quantitative trait loci (QTL). He was homozygous for SNP rs7482144 C>T minor allele (Xmn I polymorphism) at 158 bp 5' to the Gγ-globin gene on chr 11p15; homozygous for rs9399137 minor allele in the HBSIL-MYB intergenic polymorphism (HMIP) on chr 6q23, homozygous for the 3-bp deletion which is in linkage disequilibrium with rs9399137 minor allele [Farrell et al, Blood 117:4935, 2011]; and heterozygous for rs766432 minor allele in the 2nd intron of BCL11A on chr 2p16. Thus he had alleles associated with elevated HbF in all 3 QTL. No mutation was found in his KLF1 genes. Within HS2 in his β LCR is the motif (TA)9 (CA)2 (TA)2 CG (TA)10 which is found in Senegal β-globin haplotype 3 [Öner et al, Blood 79:813, 1992]. The Corfu deletion removes part of the δ-globin gene and ∼6 kb upstream flanking sequence, encompassing the 2-kb region reported to be necessary for γ-globin gene silencing [Sankaran et al, NEJM 365:807, 2011], and is often in LD with the IVSI-5 G>A severe β+-thalassemia mutation. Corfu heterozygotes have slightly increased HbF, but Corfu homozygotes have HbF ∼95%. We sequenced 6.5 kb upstream of δ-globin gene in our patient, and found homozygosity throughout and only 2 nucleotide differences from the GRCh37/hg19 assembly sequence: C>T (rs3759074) at 2,065 bp, and T>G (rs7948416) at 718 bp upstream of δ globin gene transcription start site. No deletion was found. At the repressor protein BP1 binding site 530 bp upstream of the β-globin gene, our patient had the common reference sequence, (AC)2 (AT)7 T7. Among 13 subjects heterozygous for codon 8 (–AA) β0-thalassemia mutation, their Hb was 11.6 ± 1.8 g/dL, HbF 2.8 ± 2.6% [Öner et al, Hemoglobin 14:1, 1990]. We studied two unrelated codon 8 (–AA) heterozygotes. One was a 37-year old woman with Hb 9.8, HbA 88%, HbF 5.7%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 6q23 and 2p16. The other was a 24-year old woman with Hb 11.2, HbA 90%, HbF 7.5%. She was homozygous for the Xmn I polymorphism, and heterozygous for the HbF QTL on chr 2p16. These results support the hypothesis that determinant(s) in cis to the β-globin gene cluster, including the Xmn I QTL and related functional motif(s), in concert with HMIP and BCL11A QTL can sustain high-level γ-globin gene transcription in adults. Robust γ-mRNA accumulation and HbF expression occur only when β-mRNA is markedly decreased due to nonsense mediated decay in the codon 8 (–AA) homozygote, as has been shown in patients homozygous for the Corfu deletion [Chakalova et al, Blood 105:2154, 2005] and in experimental model system [Russell, Eur J Haematol 79:516, 2007]. Our findings could have implications for the therapeutic design to induce HbF expression in β-hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

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