A Screen for Regulators of Globin Switching in the Zebrafish Embryo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 826-826
Author(s):  
Jared J Ganis ◽  
Elizabeth B Riley ◽  
James Palis ◽  
Leonard I. Zon
Keyword(s):  
Transcription Factors ◽  
Wnt Signaling ◽  
Wnt Pathway ◽  
Globin Gene ◽  
Ips Cells ◽  
Nuclear Hormone Receptor ◽  
Erythroid Cell ◽  
Control Group ◽  
Globin Genes ◽  
Knock Down

Abstract Abstract 826 The switching of the globin genes involves critical transcriptional regulators such as BCL11A, EKLF and SOX6, and the induction of fetal globin has been shown to ameliorate the symptoms of diseases such as sickle cell anemia. Recently, there has been interest in driving iPS cells to produce mature red cells that express adult globin genes in an attempt to make these cells therapeutically useful. Here, to understand hemoglobin switching and the molecular pathways that allow the establishment of an adult fate in embryonic tissues, we utilized a screening approach in the zebrafish model. The concept of the screen is to find transcription factors that are expressed in a stage-specific manner, and manipulate the expression of these genes to alter the cell fate of embryonic erythroid cells. In order to generate a candidate list of genes, microarray analysis was performed on murine yolk sac, fetal liver and adult derived red blood cells and red blood cell precursors, which express unique sets of globin genes. Pair-wise comparison of these populations yielded 879 unique differentially regulated genes. GO term analysis was used to narrow the list to 49 transcription factors. We focused on the transcription factors that might increase adult globin expression in the embryo based on their differential expression in the microarrays. Morpholinos were used to knock down these 24 genes by individually injecting each into one-cell stage embryos, allowing the embryos to reach 24 hpf and performing in situ hybridization for the adult globin gene αa1. The number of adult globin positive cells present in each embryo was counted for a clutch control group, which on average has 2–4 positive cells per embryo, and three doses of morpholino. We identified 4 genes, Tcf7l2, Ncoa1, Hif1al and E2F5, the knock down of which results in a significant increase in the number of adult globin positive cells in at least one dose of morpholino (control [n=53, mean=6.34], 6ng [n=56, mean=15.07], p=<0.0001; control [n=35, mean=1.543], 4ng [n=56, mean=2.75], p=<0.01; control [n=19, mean=1.368], 12ng [n=16, mean=6.188], p=<0.0001; control [n=44, mean=1.091], 4ng [n=30, mean=2.7], p=<0.05, respectively). Pair-wise knock down of these genes were also tested, and the combinations of Ncoa1 and E2F5, Tcf7l2 and E2F5 and Tcf7l2 and Ncoa1 were found to synergistically increase the number of adult globin expressing cells (control [n=49, mean=0.5306], knock down [n=38, mean=9.895], p=<0.0001; control [n=49, mean=7.633], knock down [n=54, mean=17.41], p=<0.0001; control [n=20, mean=2.95], knock down [n=28, mean=too numerous to count], p=<0.0001, respectively). The combined knock down of Tcf7l2 and Ncoa1 was both the strongest inducer of adult globin expression and had the lowest toxicity of the pair-wise combinations. Further characterization of this phenotype shows that, while many globin genes are up regulated, both of the adult globin genes, αa1 and βa1, are upregulated to a higher degree than other globin genes. In order to determine if the Wnt pathway is responsible for phenotype observed with the Tcf7l2 morpholino, we tested the Wnt pathway inhibitors IWR1 and XAV939. Both drugs phenocopied the Tcf7l2 knockdown response. In addition, XAV939 synergies with the Ncoa1 morpholino to enhance the increase in adult globin observed in a similar manner to Tcf7l2 knockdown. These results indicate that modulation of Wnt signaling, rather than a Wnt-independent function of Tcf7l2, is responsible for the phenotype and regulation of globin gene expression. Chip-Seq analysis of Ncoa1 occupancy in the erythroid cell line K562 was performed to examine potential mechanisms of action. Significant binding was observed at the enhancers of the α- and β-globin loci, indicating that the nuclear hormone receptor pathway may be acting directly on the globin loci to modulate globin expression patterns. These results indicate that Wnt signaling in combination with alterations of other pathways regulated by Ncoa1 are responsible for stage-specific globin expression. Our studies have impact on the understanding of globin switching in vertebrates, and could establish new methods to activate specific globins clinically, and to make iPS cells form adult-type tissues. Disclosures: Zon: Fate Therapeutics: Founder Other.

scholarly journals Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression

2018 ◽  
Vol 38 (19) ◽  
Author(s):  
Yong Shen ◽  
MacLean A. Bassett ◽  
Aishwarya Gurumurthy ◽  
Rukiye Nar ◽  
Isaac J. Knudson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Chromosome 11 ◽  
Erythroleukemia Cell ◽  
High Level ◽  
Globin Gene Expression

ABSTRACT The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5′ end, followed by the two fetal γ-globin genes, and with the adult β- and δ-globin genes being located at the 3′ end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.

scholarly journals Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor

2009 ◽  
Vol 29 (21) ◽  
pp. 5900-5910 ◽  
Author(s):  
Shermi Y. Liang ◽  
Babak Moghimi ◽  
Valerie J. Crusselle-Davis ◽  
I-Ju Lin ◽  
Michael H. Rosenberg ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transgenic Mice ◽  
Rna Polymerase Ii ◽  
Globin Gene ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Dominant Negative Mutant ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Transcription Complexes

ABSTRACT Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and β-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all β-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the β-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71+ progenitor and Ter-119+ mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.

scholarly journals Regulation of Wnt Signaling by FOX Transcription Factors in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3446
Author(s):  
Stefan Koch
Keyword(s):  
Transcription Factors ◽  
Wnt Signaling ◽  
Wnt Pathway ◽  
Treatment Options ◽  
Wnt Signaling Pathway ◽  
Tissue Homeostasis ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Forkhead Box ◽  
Signaling Activity

Aberrant activation of the oncogenic Wnt signaling pathway is a hallmark of numerous types of cancer. However, in many cases, it is unclear how a chronically high Wnt signaling tone is maintained in the absence of activating pathway mutations. Forkhead box (FOX) family transcription factors are key regulators of embryonic development and tissue homeostasis, and there is mounting evidence that they act in part by fine-tuning the Wnt signaling output in a tissue-specific and context-dependent manner. Here, I review the diverse ways in which FOX transcription factors interact with the Wnt pathway, and how the ectopic reactivation of FOX proteins may affect Wnt signaling activity in various types of cancer. Many FOX transcription factors are partially functionally redundant and exhibit a highly restricted expression pattern, especially in adults. Thus, precision targeting of individual FOX proteins may lead to safe treatment options for Wnt-dependent cancers.

scholarly journals Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming

Blood ◽  
2009 ◽  
Vol 114 (11) ◽  
pp. 2299-2306 ◽  
Author(s):  
Orapan Sripichai ◽  
Christine M. Kiefer ◽  
Natarajan V. Bhanu ◽  
Toshihiko Tanno ◽  
Seung-Jae Noh ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Protein Expression ◽  
Histone Modification ◽  
Globin Gene ◽  
Expression Patterns ◽  
Fetal Hemoglobin ◽  
Transcriptome Profiling ◽  
Globin Genes

Abstract Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 365-365 ◽  
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.

Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽  
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.

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.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

Abstract The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

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

2001 ◽  
Vol 21 (1) ◽  
pp. 73-80 ◽  
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.

Functional Characterization of a Dnase I Hypersensitive Site Located 4Kbp Upstream of the Gγ-Globin Gene Using a Synthetic Zinc Finger DNA-Binding Domain

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 320-320
Author(s):  
Yong Shen ◽  
Mir Hossain ◽  
Isaac Knudson ◽  
Shaleen Thakur ◽  
Jorg Bungert
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Western Blotting ◽  
Chromatin Immunoprecipitation ◽  
Cell Clone ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Single Cell Clone ◽  
Empty Vector

Abstract The human β-globin genes are expressed in a developmental stage-specific manner and regulated by many cis- and trans-regulatory components including a locus control region (LCR) and proximal promoter and enhancer elements. The two γ-globin genes, Gγ and Aγ, are expressed in the fetal period. Persistent expression of γ-globin in the adult ameliorates the symptoms associated with mutations of the adult β-globin gene, such as sickle cell disease or β-thalassemia. Thus, understanding the mechanisms by which the fetal globin genes are activated and silenced during development may lead to new avenues for the treatment of hemoglobinopathies. Using data from the human ENCODE project we identified a DNase I hypersensitive site located 4 Kbp upstream of the Gγ-globin gene (Gγ -4Kb DHS) in K562 cell line. Gγ -4Kb DHS is characterized by the presence of histone modifications typical for enhancer elements (H3K4 monomethylation and H3K27 acetylation) and binding of ubiquitous (USF, E2F, YY1, c-Myc, Egr1, and MafK) or tissue-restricted (NF-E2) transcription factors in K562 cells which express high levels of the γ-globin genes (Figure 1). The presence of USF and NF-E2 is interesting as both proteins have been implicated in the recruitment of transcription complexes to the β-globin gene locus (Crusselle-Davis et al., 2006, Mol. Cell. Biol.; Liang et al., 2009 Mol. Cell. Biol.; Zhou et al., 2010, J. Biol. Chem.; Stee & Hossain et al., 2015, Mol. Cell. Biol.). We generated and expressed in K562 cells a synthetic Zinc Finger DNA-Binding Domain (ZF-DBD) designed to specifically target the Gγ -4Kb DHS and interfere its activities (ZF@Gγ-4KbDHS). The target site for the ZF-DBD overlaps with a CCCAC Egr1 motif and is close to an E-box sequence, which is predicted to bind USF and c-Myc (Figure 1). Figure 2A and C shows the Western blot results for cells transfected with empty vector or cells expressing the ZF@Gγ-4KbDHS in cell pools or a single cell clone selected from the pool of transfected cells (Figure 2A and C, respectively). We analyzed the binding of ZF@Gγ-4KbDHS at the globin locus in K562 cells using Chromatin Immunoprecipitation (ChIP). The data demonstrate that the ZF@Gγ-4KbDHS efficiently interacted with the Gγ -4Kb DHS and less efficiently with the γ-globin promoters (Figure 2B). Expression of the ZF@Gγ-4KbDHS led to a significant reduction in expression of the γ-globin genes but had no effect on expression of the GATA-1 gene (Figure 2D). The data suggest that the Gγ -4Kb DHS contributes to high-level γ-globin gene expression in K562 cells. Additionally, a SNP (rs11036496, Figure 1) within the Gγ -4Kb DHS has been reported to be associated with a disorder of γ-globin gene expression in African Americans and Chromatin Conformation Capture (3C) experiments showed that the Gγ-globin upstream region participates in interactions with the LCR and γ-globin genes (Shriner et al., 2015, BMC Medical Genetics; Kiefer et al., 2011, Blood). Therefore, further characterization of the Gγ -4Kb DHS will enhance understanding molecular mechanism(s) regulating hemoglobin switching. Figure 1 Epigenetic signatures and transcription factor binding at the Gγ -4Kb DHS. Shown on top are the two γ-globin genes and the relative position of the Gγ -4Kb DHS. Shown on the bottom is an enlarged view of the Gγ -4Kb DHS and binding peaks for several transcription factors as indicated. Figure 1. Epigenetic signatures and transcription factor binding at the Gγ -4Kb DHS. Shown on top are the two γ-globin genes and the relative position of the Gγ -4Kb DHS. Shown on the bottom is an enlarged view of the Gγ -4Kb DHS and binding peaks for several transcription factors as indicated. Figure 2 Reduced expression of γ-globin in K562 cells expressing the ZF@Gγ-4KbDHS. K562 cells were transfected with a plasmid expressing the ZF@Gγ-4KbDHS or with an empty vector. The K562 cells (pool) were subjected to Western blotting (A) and to Chromatin Immunoprecipitation (ChIP) using antibodies specific for the FLAG-tag, which is linked to the ZF@Gγ-4KbDHS, or negative control antibodies IgG (B). Single clonal K562 cells were subjected to Western blotting using antibodies specific for the ZF-DBD backbone (αZF) or for CTCF or GATA-1 as indicated (C). The single cell clone expressing the ZF@Gγ-4KbDHS was subjected to expression analysis by RT-qPCR using primers specific for γ-globin or GATA-1 as indicated (D). Figure 2. Reduced expression of γ-globin in K562 cells expressing the ZF@Gγ-4KbDHS. K562 cells were transfected with a plasmid expressing the ZF@Gγ-4KbDHS or with an empty vector. The K562 cells (pool) were subjected to Western blotting (A) and to Chromatin Immunoprecipitation (ChIP) using antibodies specific for the FLAG-tag, which is linked to the ZF@Gγ-4KbDHS, or negative control antibodies IgG (B). Single clonal K562 cells were subjected to Western blotting using antibodies specific for the ZF-DBD backbone (αZF) or for CTCF or GATA-1 as indicated (C). The single cell clone expressing the ZF@Gγ-4KbDHS was subjected to expression analysis by RT-qPCR using primers specific for γ-globin or GATA-1 as indicated (D). Disclosures No relevant conflicts of interest to declare.

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