Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity.

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Characterization of Adult α- and β-Globin Genes in the Zebrafish

Blood ◽

10.1182/blood.v89.2.688 ◽

1997 ◽

Vol 89 (2) ◽

pp. 688-700 ◽

Cited By ~ 71

Author(s):

Fung-Yee Chan ◽

Judith Robinson ◽

Alison Brownlie ◽

Ramesh A. Shivdasani ◽

Adriana Donovan ◽

...

Keyword(s):

High Performance ◽

Globin Gene ◽

Regulatory Elements ◽

Reversed Phase ◽

Cdna Clones ◽

Globin Genes ◽

Erythroid Cells ◽

Insight Into ◽

Globin Gene Expression

Abstract Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major α globins and two β globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult α- and β-globin cDNA clones that encode some of these globins. High levels of α- and β-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the α- and β-globin genes are tightly linked on the same chromosome and are arrayed in a 3′-5′ to 5′-3′ configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.

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Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells

Blood ◽

10.1182/blood-2012-11-466227 ◽

2013 ◽

Vol 121 (17) ◽

pp. 3493-3501 ◽

Cited By ~ 45

Author(s):

Maria Amaya ◽

Megha Desai ◽

Merlin Nithya Gnanapragasam ◽

Shou Zhen Wang ◽

Sheng Zu Zhu ◽

...

Keyword(s):

Gene Expression ◽

Major Part ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Cells ◽

Key Points ◽

Globin Gene Expression ◽

Partial Depletion

Key Points Mi2β exerts a major part of its silencing effect on embryonic and fetal globin genes by positively regulating the BCL11A and KLF1 genes. Partial depletion of Mi2β induces increased γ-globin gene expression in primary human erythroid cells without impairing differentiation.

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Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽

10.1182/blood.v65.3.705.705 ◽

1985 ◽

Vol 65 (3) ◽

pp. 705-712 ◽

Cited By ~ 8

Author(s):

NP Anagnou ◽

TY Yuan ◽

E Lim ◽

J Helder ◽

S Wieder ◽

...

Keyword(s):

Gene Expression ◽

Human Chromosome ◽

Globin Gene ◽

Globin Genes ◽

Chromosome 16 ◽

Regulatory Factors ◽

Erythroleukemia Cells ◽

Erythroleukemia Cell ◽

Globin Gene Expression ◽

Mouse Erythroleukemia

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

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Ham-Wasserman Lecture

Hematology ◽

10.1182/asheducation-2004.1.1 ◽

2004 ◽

Vol 2004 (1) ◽

pp. 1-13 ◽

Cited By ~ 12

Author(s):

Douglas R. Higgs

Keyword(s):

Gene Expression ◽

Genetic Disease ◽

Globin Gene ◽

Hematopoietic Cells ◽

Current Understanding ◽

Globin Genes ◽

Human Genetic Disease ◽

The Past ◽

Inherited Mutations ◽

Globin Gene Expression

Abstract Over the past fifty years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the α and β globin genes are normally regulated and documenting the effects of inherited mutations which cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found with increasing frequency. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression which, in turn, may eventually open up new avenues for improving the management of patients with common types of thalassemia.

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Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.1210.1210 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1210-1210

Author(s):

Lauren Sterner ◽

Toru Miyazaki ◽

Larry Swift ◽

Ann Dean ◽

Jane Little

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Fetal Liver ◽

Globin Gene ◽

Short Chain Fatty Acids ◽

Yolk Sac ◽

Globin Genes ◽

Wild Type ◽

Alpha Type ◽

Globin Gene Expression

Abstract We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels <1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n>=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p<.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress. Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs % Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p < .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p < .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p < .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns

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Subdomains of the Baboon (P. anubis) β-Globin Gene Cluster Are Differentially Sensitive to Dacogen Treatment.

Blood ◽

10.1182/blood.v106.11.525.525 ◽

2005 ◽

Vol 106 (11) ◽

pp. 525-525

Author(s):

Janet Chin ◽

Donald Lavelle ◽

Bryan Roxas ◽

Kestis Vaitkus ◽

Maria Hankewych ◽

...

Keyword(s):

Gene Expression ◽

Histone Acetylation ◽

Intergenic Region ◽

Globin Gene ◽

Globin Genes ◽

Gene Complex ◽

Alu Sequence ◽

Low Levels ◽

Globin Promoter ◽

Globin Gene Expression

Abstract Understanding the mechanism responsible for the developmental regulation of the β-like globin genes would be important in the design of future pharmacologic therapies to increase fetal hemoglobin (HbF) in patients with sickle cell disease and β-thalassemia. The baboon is a valuable and relevant experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to human, and HbF levels are greatly increased following treatment of baboons with the DNA methyltransferase inhibitor Dacogen (5-aza-2′-deoxycytidine; DAC). To investigate the relationship between DNA methylation, chromatin structure and globin gene expression, the pattern of acetylated histone H3 (ac-H3) and H4 (ac-H4) within the β-globin gene complex was compared in purified erythroblasts from baboon fetal liver (FL; n=2) and bone marrow (ABM; n=2) of adult baboons pre and post DAC treatment. HbF increased to high levels (67.8%, 61.9%) in respective animals and methylation of 18 CpG sites within the ε- and γ globin genes was reduced >50% following DAC treatment. Enrichment of ac-H3 and ac-H4 throughout the β-globin gene complex was measured by chromatin immunoprecipitation (ChIP) followed by real time PCR. In FL, equivalent levels of ac-H3 and ac-H4 were observed near the ε-globin and γ-globin promoters that were 3 fold higher than near the Aγ-enhancer and pseudo-β gene and 5–14 fold higher than near the β-globin promoter. In pretreatment ABM, levels of ac-H3 and ac-H4 near the β-globin promoter were 4–6 fold greater than near the γ-globin promoter, Aγ-enhancer, and pseudo-β gene and 10-15 fold higher than near the ε-globin promoter. The lowest levels of histone acetylation were observed in a 6kb subdomain within the γ-β intergenic region extending from the duplicated Alu sequence to 3′ of the δ-globin gene. Following DAC treatment, histone acetylation of the ε-, γ-, and pseudo-β genes and Aγ-enhancer increased 4-10 fold, while histone acetylation of the β-globin gene remained unchanged. This resulted in equivalent levels of histone acetylation associated with the γ-globin gene, Aγ-enhancer, pseudo-β-, and β-globin genes that were 3 fold greater than with the ε-globin gene. The levels of histone acetylation within the 6 kb subdomain of the γ-β intergenic region remained low. Our results suggest that three subdomains of chromatin are present within the baboon β-globin gene complex. One subdomain that encompasses the ε-, γ-, and pseudo-β genes is characterized by high levels of histone acetylation in FL and low levels in ABM. DAC treatment increases histone acetylation within this region to levels observed near the β-globin gene. A second subdomain near the β-globin gene is characterized by high levels of histone acetylation in ABM and low levels in FL. Histone acetylation of the β-globin gene within this subdomain remains high following DAC. A third subdomain found within the γ-β intergenic region surrounding the duplicated Alu sequences is characterized by a low level of histone acetylation in both FL and ABM. The level of histone acetylation of this region remains low following DAC. We conclude that three chromatin subdomains within the β-globin gene complex are differentially sensitive to DAC-induced changes in histone acetylation.

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Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Cell Disease ◽

Globin Mrna ◽

Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

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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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Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽

10.1182/blood.v110.11.4102.4102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4102-4102

Author(s):

Vladan P. Cokic ◽

Bojana B. Beleslin-Cokic ◽

Constance Tom Noguchi ◽

Alan N. Schechter

Keyword(s):

Progenitor Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

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Transcriptional Regulation of Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.sci-7.sci-7 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-7-SCI-7

Author(s):

Mitchell J. Weiss

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Transcriptional Activation ◽

Zinc Finger Protein ◽

Conflicts Of Interest ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Development ◽

Globin Gene Expression

Abstract Abstract SCI-7 Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas. Disclosures No relevant conflicts of interest to declare.

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