New Strategies to Define Regulators of Fetal Hemoglobin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-17-SCI-17
Author(s):  
Stuart H. Orkin
Keyword(s):  
High Throughput Screening ◽  
Genome Wide Association Study ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Globin Genes ◽  
Chromosome 2 ◽  
Erythroid Cells ◽  
Hemoglobin F ◽  
Developmental Switches

Abstract Abstract SCI-17 The beneficial effect of hemoglobin F (HbF, α2γ2) expression on the phenotypes of sickle cell disease and the β-thalassemias has been recognized for many years. Therefore, retardation of the fetal-to-adult (HbF to HbA, α2b2) switch or reactivation of g-globin expression in erythroid cells of adults has been a long sought goal. Despite molecular analysis of the human β-globin gene cluster and intense study of transgenic mice harboring human globin genes, the nuclear factors mediating hemoglobin switching and maintaining γ-globin gene silencing have remained unknown. The situation has changed recently with the success of new genetic tools, including genome-wide association study (GWAS), in the identification of candidate gene regions in which genetic variation correlates with HbF levels. Three genomic regions have been fingered: the β-globin cluster itself, the BCL11A gene on chromosome 2, and the HB1SL-Myb region on chromosome 6. Taken together, these regions account for ∼40% of the variation in HbF levels. Functional studies have shown that BCL11A encodes a dose-sensitive, zinc-finger repressor that is required for maintenance of silencing of γ-globin in adult erythroid cells. BCL11A also controls the developmental switches from embryonic to adult β-like globins in the mouse and fetal to adult globin in human transgenes harbored in the mouse. BCL11A binds downstream of the γ-globin gene and in the locus control region, but not at the γ-promoters. Mechanistic studies reveal that BCL11A cooperates with another factor, SOX6, that binds within the γ-promoters. The identification of genetically validated regulators has therapeutic implications for directed reactivation of HbF in adults. For stage-specific repressors, such as BCL11A, inhibition of their expression, or interference with their function or interactions with other regulators, could constitute new therapeutic strategies. While transcription factors have been generally considered “undruggable”, advances in high-throughput screening of complex libraries of small molecules and in the use of RNA interference in vivo may provide new platforms for discovery and refinement of HbF inducers. The presentation will illustrate some of these potential approaches. Disclosures: No relevant conflicts of interest to declare.

Transcriptional Regulation of Erythropoiesis.

Blood ◽  
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.

Control of Hemoglobin Switching by BCL11A.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5-5
Author(s):  
Jian Xu ◽  
Vijay G. Sankaran ◽  
Yuko Fujiwara ◽  
Stuart H. Orkin
Keyword(s):  
Gene Silencing ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Association Studies ◽  
Globin Gene ◽  
Clinical Severity ◽  
Genome Wide Association Studies ◽  
Erythroid Cells ◽  
B Locus

Abstract Abstract 5 All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

Controlling Long-Range Genomic Interactions to Reprogram the β-Globin Locus

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 280-280
Author(s):  
Wulan Deng ◽  
Jeremy W Rupon ◽  
Hongxin Wang ◽  
Andreas Reik ◽  
Philip D. Gregory ◽  
...  
Keyword(s):  
Long Range ◽  
Globin Gene ◽  
Chromatin Interaction ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Distal Enhancer ◽  
Adult Type ◽  
High Level ◽  
Target Promoters

Abstract Abstract 280 Distal enhancers physically contact target promoters to confer high level transcription. At the mammalian β-globin loci long-range chromosomal interactions between a distal enhancer, called the locus control region (LCR), and the globin genes are developmentally dynamic such that the LCR contacts the embryonic, fetal and adult globin genes in a stage-appropriate fashion. LCR-globin gene interactions require the nuclear factor Ldb1. Recently, we employed artificial zinc finger (ZF) proteins to target Ldb1 to the endogenous β-globin locus to force an LCR-promoter interaction. This led to substantial activation of β-globin transcription and suggested that forced chromatin looping could be employed as a powerful tool to manipulate gene expression in vivo (Deng et al., Cell 2012). Reactivation of the fetal globin genes in adult erythroid cells has been a long-standing goal in the treatment of patients with sickle cell anemia. Therefore, building on our findings, we investigated whether the developmentally silenced embryonic globin gene βh1 can be re-activated in adult murine erythroblasts by re-directing the LCR away from the adult type globin gene and towards its embryonic counterpart. To this end, Ldb1 was fused to artificial ZF proteins (ZF-Ldb1) designed to bind to the βh1 promoter. ZF-Ldb1 was introduced into definitive erythroid cells in which only the adult but not the embryonic β-like globin gene is expressed. In vivo binding of the ZF-Ldb1 to its intended target was verified by chromatin immunoprecipitation assay. Strikingly, expression of ZF-Ldb1 re-activated βh1 transcription up to approximately ∼15% of total cellular β-globin production. This suggests that forced tethering of a looping factor to a select promoter can be employed to override a pre-existing developmental long-range chromatin interaction to reprogram a developmentally controlled gene locus. We are now in the process of testing whether our approach might be suitable to reactivate the silent fetal globin genes in adult human erythroid cells. These studies are underway and the results will be discussed at the meeting. Disclosures: Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.

scholarly journals KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic β-like globin genes in vivo

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2566-2571 ◽  
Author(s):  
Priyadarshi Basu ◽  
Pamela E. Morris ◽  
Jack L. Haar ◽  
Maqsood A. Wani ◽  
Jerry B. Lingrel ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Wild Type ◽  
Evolutionarily Conserved ◽  
Primitive Erythropoiesis ◽  
Globin Gene Expression

AbstractThe Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the β-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the β-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and βh1-globin but not ζ-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult βmaj- and βmin-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic ϵ-globin gene but not the adult β-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death. (Blood. 2005;106: 2566-2571)

Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1536-1542
Author(s):  
LJ Burns ◽  
JG Glauber ◽  
GD Ginder
Keyword(s):  
Sodium Butyrate ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Hemoglobin Switching ◽  
High Level

An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

scholarly journals The role of the polycomb complex in silencing α-globin gene expression in nonerythroid cells

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3889-3899 ◽  
Author(s):  
David Garrick ◽  
Marco De Gobbi ◽  
Vasiliki Samara ◽  
Michelle Rugless ◽  
Michelle Holland ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Globin Gene ◽  
Cpg Islands ◽  
Gene Activation ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Polycomb Complex ◽  
Globin Gene Expression

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human α- and β-globin genes are silenced by fundamentally different mechanisms. The α-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the β-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the α-cluster by sequences within the CpG islands associated with their promoters; the β-globin promoters do not lie within such islands. Chromatin associated with the α-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The α- and β-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.

scholarly journals Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1536-1542 ◽  
Author(s):  
LJ Burns ◽  
JG Glauber ◽  
GD Ginder
Keyword(s):  
Sodium Butyrate ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Hemoglobin Switching ◽  
High Level

Abstract An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

scholarly journals In Human Beta-Globin Gene Locus, ERV-9 LTR Retrotransposon Interacts with and Activates Beta- but Not Gamma-Globin Gene

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2686-2686
Author(s):  
Dorothy Tuan ◽  
Wenhu Pi
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Locus ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Endogenous Retroviruses ◽  
Ltr Retrotransposon ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Primary Target ◽  
Bac Clone

Abstract Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) comprise over 40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions was not known. In the human b-globin gene locus, an ERV-9 LTR retrtransposon is located near the 5’ end of the locus control region (LCR) at 40-70 kb upstream of the human fetal g- and adult b-globin genes. To address the functional significance of the intergenic ERV-9 LTR, we generated transgenic (Tg) mice carrying a 100 Kb BAC clone spanning the entire human b-globin gene locus from the ERV-9 LTR to b-globin gene and showed that the LTR retrotransposon serves long-range, beneficial host function (Pi et al., PNAS 2010): The ERV-9 LTR containing multiple CCAAT and GATA motifs competitively recruits high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. Deletion of the ERV-9 LTR by Cre-loxP mediated in situ recombination in the BAC Tg mice suppresses transcription of b-globin gene but activates transcription of g-globin gene. The results indicate that the ERV-9 LTR activates transcription of b-globin gene in erythroid cells during development. Therefore, LTR deletion drastically suppressed b-globin gene and re-activated g-globin gene through a competitive mechanism of globin gene switching. Alternatively, the primary effect of the ERV-9 LTR could be to suppress g-globin gene during development. Therefore, LTR deletion re-activated g-globin gene, which then suppressed transcription of b-globin gene. To differentiate between these two possibilities, we utilized Mx1-Cre mice to conditionally delete the ERV-9 LTR in the erythroid cells of the adult BAC transgene mice, in which g-globin gene was already silenced and b-globin gene fully activated. If the primary target of the ERV-9 LTR enhancer complex was g-globin gene, deletion of the ERV-9 LTR should not be able to activate the already silenced g-globin gene nor to suppress the fully active b-globin gene. However, the same effect on transcriptional suppression of b-globin gene and re-activation of g-globin gene was observed. These results indicate that the primary target of the ERV-9 LTR is the b-globin gene and not the g-globin gene. The molecular factors underlying the preferential interaction between the ERV-9 LTR and the b-globin gene are under investigation and will be presented Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 121 (17) ◽  
pp. 3493-3501 ◽  
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.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

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