Smarca5 Regulates Ctcf Recruitment to Chromatin, Including to Regulatory Loci Involved In Control of Globin Gene Expression In Erythroleukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5159-5159
Author(s):  
Martina Kapalova ◽  
Juraj Kokavec ◽  
Nikola Curik ◽  
Pavel Burda ◽  
Arthur I. Skoultchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Protein Levels ◽  
Genetic Imprinting ◽  
Regulatory Loci ◽  
Globin Gene Expression

Abstract Abstract 5159 Transcription factor Ctcf (CCCTC-binding factor) represents a major regulatory component of epigenetic regulation by recognizing its unmethyled DNA binding sites, resulting in changes in expression of neighboring genes. Ctcf plays an important role in transgenerational genetic imprinting. Very little is known about its role in hematologic malignancies. Ctcf has been described to promote differentiation of human erythroleukemia K562 cells (Torano 2005). We studied Ctcf in mouse erythroleukemia (MEL) cells and found it is expressed at both the mRNA and protein levels. Using chromatin immunoprecipitation (ChIP), we found that Ctcf is recruited to the H19/Igf2 imprinting control region (ICR) and also to the promoters of the alpha globin genes (Hba-a1, Hba-a2) as well as the beta globin locus control region (LCR) in MEL cells. To determine the mechanism by which Ctcf interacts with chromatin, we tested its interaction with chromatin remodeling proteins that associate with these DNA targets, including the well known Imitation Switch (ISWI class) ATPase Smarca5 (Snf2h). Using coimmunopreciptiation and ChIP experiments we found that Smarca5 and Ctcf interact on DNA. Next, we used MEL cells expressing an inducible Smarca5 shRNA. Doxycycline induction of Smarca5 shRNA led to a 5-fold decrease in Smarca5 mRNA and protein levels within 48hrs. ChIP experiments demonstrated that depletion of Smarca5 was accompanied by loss of Ctcf from the aforementioned loci indicating Ctcf requires Smarca5 for its association with chromatin. Furthermore, this was followed by significantly decreased levels H19 RNA. Our data provide evidence that Smarca5 regulates Ctcf recruitment to chromatin, including to regulatory loci involved in controlling globin gene expression. (Grants # IGA 10310-3, MSMT 2B06077, GAUK 251070 45410, SVV-2010-254260507, NIH R01CA154239). 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.

O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1020-1020
Author(s):  
Kenneth R Peterson ◽  
Zhen Zhang ◽  
Ee Phie Tan ◽  
Anish Potnis ◽  
Nathan Bushue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sodium Butyrate ◽  
Yeast Artificial Chromosome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Globin Gene Expression ◽  
Dynamic Cycling

Abstract Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. 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.

Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3627-3627
Author(s):  
Elliot M. Epner ◽  
Jin Wang ◽  
Jing Huang
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Globin Genes ◽  
Phenotypic Analysis ◽  
Pol Ii ◽  
Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3200-3200
Author(s):  
Tânia Regina Zaccariotto ◽  
Daniela Maria Ribeiro ◽  
Joao Machado-Neto ◽  
Magnun N N Santos ◽  
Carolina Lanaro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Qrt Pcr ◽  
Globin Gene Expression

Abstract Abstract 3200 Background: Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P2 (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter. This work was supported by FAPESP, CNPq and INCTS. Disclosures: No relevant conflicts of interest to declare.

Homozygous Expression of a Novel Senegalese-Type Partial Deletion of the Beta and Delta Genes Causes a Delta0 Beta+ Thalassemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2128-2128
Author(s):  
Hernan Sabio ◽  
Natalia Dixon ◽  
Ferdane Kutlar ◽  
Niren Patel ◽  
Hanfang Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Analysis ◽  
Conflicts Of Interest ◽  
Clinical Phenotype ◽  
Globin Gene ◽  
African Ancestry ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Chain Imbalance ◽  
Globin Gene Expression

Abstract Abstract 2128 Clinical phenotype in β-thalassemia syndromes is determined by the degree of chain imbalance. An increase in γ-globin production will compensate for the absent or deficient β-globin synthesis and will result in the amelioration of the chain imbalance, and hence an improvement in clinical features. The known genotypes of δβ-thalassemia are associated with an increase in Hb F production, which results in the amelioration of the clinical presentation. Most δβ-thalassemias result from deletions that remove the δ- and β-globin genes, (δβ)0 with a compensatory increase in γ-globin (Hb F) expression. We report an unusual case of homozygous δ0β+ thalassemia that provides interesting insights into increased γ-globin expression and the regulation of β-globin gene expression. An 8-year old boy of African ancestry presented with lifelong jaundice and pallor. He also experienced episodes of worsening symptoms. He exhibited frontal bossing, pale mucosa, scleral icterus, and moderate splenomegaly. He was known to have G6PD deficiency and was suspected of having additional erythrocyte pathology. The CBC revealed a Hb of 8.7, Hct 26.4, MCV 64.7, WBC 10,700, platelets 283,000, reticulocytes 2.2%, and total bilirubin 5.3. Hemoglobin analysis by HPLC and IEF revealed HbA 13.4%, Hb F 86.6%, and no additional components. Alpha thalassemia −3.7kb deletion was not detected. Globin chain analysis revealed α, β, Gγ and AγI chains. DNA analysis revealed a novel Senegalese-type deletion of the beta and delta genes, resulting in a delta0 beta+ thalassemia. The subject's parents who were both from the same small village in Niger had normal hematology values. Their hemoglobin analyses revealed Hb A 94. 8%, Hb A2 2.0%, Hb F 3.2% and Hb A 93.5%, Hb A2 2.1%, Hb F 4.5% in the father and mother, respectively. They were both heterozygous for the delta-beta deletion identified in their son. DNA analysis revealed a breakpoint in the delta gene at nucleotides 54755–54760 and a breakpoint in the beta gene at nucleotides 62153– 62158 [GenBank Ref ID: HUMHBB] with a 5 nucleotide “CAACA” bp region overlapping area. The subject, who is homozygous for the identified deletions, has a clinical phenotype of thalassemia intermedia. He has not yet required red cell transfusions. This is the first instance of a Senegalese-type deletion occurring in the homozygous state. The genotype provides insights into regulation of globin gene expression. While the ∼7 Kb deletion in the δβ-intergenic region may be responsible for the increased expression of the γ-globin gene similar to Hb Lepore deletions, the continued low level expression of the β-globin gene is most probably the result of the juxtaposition of the inefficient δ-globin promoter brought in the vicinity of the β-globin gene. 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

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.

Regulation of the Human α-Globin Genes by GKLF.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1867-1867
Author(s):  
Paolo Moi ◽  
Giuseppina Maria Marini ◽  
Loredana Porcu ◽  
Isadora Asunis ◽  
Maria Giuseppina Loi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Point Mutations ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Small Interfering Rnas ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.

scholarly journals Induction of Human Fetal Globin Gene Expression by a Novel Erythroid Factor, NF-E4

2000 ◽  
Vol 20 (20) ◽  
pp. 7662-7672 ◽  
Author(s):  
Wenlai Zhou ◽  
David R. Clouston ◽  
Xi Wang ◽  
Loretta Cerruti ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Dimerization Domain ◽  
Preferential Expression ◽  
Globin Gene Expression

ABSTRACT The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human γ-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the γ promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

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.

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