Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator

Abstract A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD. Disclosures Makala: Georgia Regents University: Employment.

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Induction of Fetal HemoglobinIn VivoMediated by a Syntheticγ-Globin Zinc Finger Activator

Anemia ◽

10.1155/2012/507894 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 19

Author(s):

Flávia C. Costa ◽

Halyna Fedosyuk ◽

Renee Neades ◽

Johana Bravo de Los Rios ◽

Carlos F. Barbas ◽

...

Keyword(s):

Gene Expression ◽

Zinc Finger ◽

Yeast Artificial Chromosome ◽

Bone Marrow Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Proximal Promoter ◽

Erythroid Progenitor ◽

Globin Gene Expression

Sickle cell disease (SCD) andβ-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF,γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation ofγ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of theAγ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a humanβ-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+erythroid progenitor cells from normal donors andβ-thalassemia patients. Herein, we report that gg1-VP64 increasedγ-globin gene expressionin vivo, in peripheral blood samples from gg1-VP64β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.

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Generation of Non-Deletional Hereditary Persistence of Fetal Hemoglobin (HPFH) Beta-Yac Transgenic Mouse Models: -175 Black HPFH and -195 Brazilian HPFH

Blood ◽

10.1182/blood.v126.23.3377.3377 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3377-3377

Author(s):

Carolina A Braghini ◽

Fernando F Costa ◽

Flavia C Costa ◽

Halyna Fedosyuk ◽

Matthew Parker ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mouse ◽

Mouse Models ◽

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Wild Type ◽

Fold Enrichment ◽

Hereditary Persistence ◽

Globin Gene Expression

Abstract Fetal hemoglobin (HbF) is a major genetic modifier of the phenotypic heterogeneity in patients with the major β-globin disorders sickle cell disease (SCD) and β-thalassemia. Although the normal level of HbF postnatally is approximately 1% of total hemoglobin, some individuals have a condition known as hereditary persistence of fetal hemoglobin (HPFH), characterized by elevated synthesis of γ-globin in adulthood. HPFH is caused by small or large deletions in the β-globin locus (deletional HPFH), or point mutations in the Aγ-globin or Gγ-globin gene promoters (non-deletional HPFH). Pharmacological agents such as butyrate, decitabine, and hydroxyurea are effective in inducing HbF in vitro and in vivo. To date, hydroxyurea is the only drug approved for clinical use in sickle cell patients, although the efficacy level is variable between patients and the long-term effects of this drug remain uncertain. Therefore, current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin gene expression in adult life. Many studies have demonstrated the role of stage-specific transcription factors in β-like globin gene switching, indicating their potential as therapeutic targets in the treatment of β-hemoglobinopathies. In order to better understand the molecular pathways involved in the regulating γ-globin gene expression, we used β-YAC transgenic mice, produced with a 213 Kb β-globin locus yeast artificial chromosome, containing a 187 Kb human chromosomal insert encompassing the entire 82 Kb β-globin locus from 5'HS5 of the LCR to 3'HS1, approximately 20 Kb downstream from the β-globin gene. Four different transgenic mouse lines were included in this study: 1) wild β-YAC mice, with the normal sequence of the human β-globin locus; 2) mutant β-YAC mice with the Aγ-globin -117 G>A HPFH mutation 3) mutant β-YAC mice with the Aγ-globin -175 T>C HPFH mutation, and 4) mutant β-YAC mice with the Aγ-globin -195 T>C HPFH mutation. Adult -175 and -195 mutant β-YAC mice displayed an HPFH phenotype with an increased level of HbF. As measured by HPLC, -175 HPFH mice had the highest average level of γ-globin chains [16.4% γ/(γ+β)], followed by -195 HPFH mice (8.4%). Wild-type β-YAC control mice averaged 2.8% and -117 Greek HPFH β-YAC control mice displayed an average of 7.4%. Measurement of Aγ-globin mRNA by RNase protection analysis (RNAP) supported the HPLC data; γ/(γ+β) was 34%, 12.1%, 14.1% and less than 0.5% for -175 HPFH, -195 HPFH, -117 HPFH and wild-type β-YAC animals, respectively. Relative mRNA levels as determined by RT-qPCR were consistent with the RNAP results. Currently, we are examining our -175 and -195 HPFH mice for pancellular versus heterocellular distribution of HbF. To examine the molecular basis for the -175 and 195 HPFH phenotypes, fetal livers of these animals were collected on day E18 of gestation, after the fetal-to-adult β-like globin switch occurred, for chromatin immunoprecipitation (ChIP) analysis of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2 and LDB1. Previous unpublished DNA-protein array and ChIP data, comparing human primary erythroid cell cultures from normal donors and -195 HPFH individuals, showed a 6-fold enrichment of YY1 recruitment to the -195 region of the normal Aγ-globin promoter and a 5-fold enrichment of PAX1 recruitment to the HPFH mutant promoter, suggesting that YY1 may act as an A γ-globin gene repressor and PAX1 may be an activator when the -195 mutation is present. Preliminary ChIP experiments in β-YAC mice showed a similar pattern with YY1 enriched 2-fold in wild-type mice and PAX1 enriched 2-fold in -195 HPFH animals. Regarding -175 HPFH and wild-type β-YAC samples, we found occupancy enrichment of LMO2, TAL1 and LDB1 proteins (1.5-fold, 9-fold and 2.5-fold, respectively) in the -175 region of the Aγ-globin gene promoter in -175 HPFH β-YAC mice. Recently published studies in cell lines have shown that these three proteins form a complex with GATA-1 to mediate long-range interactions between the LCR and β-like globin genes. These mouse models provide additional tools for studying the regulation of γ-globin gene expression and may reveal new targets for selectively activating HbF. Disclosures No relevant conflicts of interest to declare.

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O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽

10.1182/blood.v122.21.1020.1020 ◽

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.

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The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.2019.2019 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2019-2019

Author(s):

Kenneth R Peterson ◽

Halyna Fedosyuk ◽

Flavia C Costa

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Wild Type ◽

Repressor Complex ◽

Definitive Erythropoiesis ◽

Gata Motif ◽

Globin Gene Expression

Abstract Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea

Blood ◽

10.1182/blood.v87.4.1604.bloodjournal8741604 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1604-1611 ◽

Cited By ~ 36

Author(s):

ZH Lu ◽

MH Steinberg

Keyword(s):

Gene Expression ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Regulatory Sequences ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Globin Gene Expression

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta- globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5′ to the beta-globin gene, a 4-bp deletion 5 to the A gamma T gene, promoters of both gamma-globin genes, 5′ flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

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Induction of Human γ Globin Gene Expression by Histone Deacetylase Inhibitors.

Blood ◽

10.1182/blood.v108.11.1583.1583 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1583-1583

Author(s):

Hua Cao

Keyword(s):

Gene Expression ◽

Histone Deacetylase ◽

Hydroxamic Acid ◽

Globin Gene ◽

Hdac Inhibitors ◽

Fetal Hemoglobin ◽

Erythroid Progenitor ◽

Globin Gene Expression ◽

Hydroxamic Acid Derivatives

In previous studies we have showed that HDAC inhibitors including hydroxamic acid derivatives of short chain fatty acids butyryl hydroxamate, propionyl hydroxamate, subericbis hydroxamic acid (SBHA), and suberoylanilide hydroxamic acid (SAHA), are potent inducers of γ globin gene expression in in vitro luciferase assays and in cultures of human adult erythroid progenitor cells. In this present study, we used μLCR Aγ transgenic mice to test whether these compounds can also induce γ gene expression in vivo. We found that in addition to γ gene induction these compounds have considerable erythropoiesis activity. Thus, Propionyl and butyryl hydroxamate increased reticulocytes of mice by 71% and 139%, the in vivo BFUe counts by 75% and 51% and the in vivo γ gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. We conclude that Hydroxamic acid derivatives can stimulate both the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. Cyclic depsipeptide FK228 is a highly potent histone deacetylase inhibitor, currently in clinical trials in cancer patients. We investigated whether FK228 also functions as inducer of human γ globin gene expression and compared Hb F induction by FK228 to that of four other HDAC inhibitors, including hydroxamic acids (TSA), synthetic benzamides (MS-275), and two cyclic tetrapeptides, Apicidin and HC-Toxin. Our results showed that FK228 is the most potent fetal hemoglobin inducer among all the HDAC inhibitors tested in our laboratory. In a concentration of 0.84 nanomolar, FK228 induces γ gene promoter activity in the dual luciferase assay by 7.81 fold. In the human erythroid progenitor cell cultures it increases the levels of γ mRNA by 8.48 fold in a concentration of 0.143 nM. In contrast, fetal hemoglobin induction by other HDAC inhibitors is achieved in concentrations that are 100 to 1000 fold higher. We conclude that FK228 is a promising compound for induction of Hb F in patients with sickle cell disease and thalassemia.

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A Mutation in a GATA-1 Binding Site 5′ to the Gγ-Globin Gene (nt -567, T>G) May Be Associated with Increased Levels of Fetal Hemoglobin.

Blood ◽

10.1182/blood.v104.11.500.500 ◽

2004 ◽

Vol 104 (11) ◽

pp. 500-500 ◽

Cited By ~ 1

Author(s):

H.Y. Luo ◽

D. Mang ◽

G.P. Patrinos ◽

C.J.Y. Wu ◽

S.H. Eung ◽

...

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Family Members ◽

Point Mutations ◽

Fetal Hemoglobin ◽

Proximal Promoter ◽

Globin Genes ◽

Nucleotide Polymorphisms ◽

Mobility Shift ◽

Globin Gene Expression

Abstract The identification of single nucleotide polymorphisms (SNPs) associated with increased fetal hemoglobin (Hb F) levels in adults provide important insights to the regulation of γ-globin gene expression, and the modulation of Hb F production in severe β hemoglobinopathies. Fourteen known point mutations located between nucleotide (nt) −110 to −205 5′ to the Gγ- and Aγ-globin genes are associated with hereditary persistence of fetal hemoglobin (HPFH). These likely affect the interactions between transcription factors and proximal promoter elements. We investigated both parents and 2 sons of an Iranian-American family, in whom the father and his younger son had elevated Hb F levels (See table). The mother and her older son were heterozygous for the (−α3.7) single α-globin gene deletion, likely accounting for their borderline microcytosis. Extensive nt sequencing of the β-globin gene and promoters of Gγ- and Aγ-globin genes was carried out. No known β-thalassemia mutation was detected in any of the 4 family members. None of the known HPFH point mutations was present. The C>T SNP (Xmn I) at nt −158 5′ to the Gγ-globin gene that has been associated with increased Gγ-globin gene expression was also not found. However, a novel T>G substitution was detected at nt −567 5′ to the Gγ-globin gene in the father and his younger son, but not in the mother and her older son. This SNP alters a putative GATA-1 binding sequence, AGATAA to AGAGAA. Haplotyping of the Gγ Aγβ region in the family showed that the T>G SNP in the father and his younger son resides on the same GγAγβ haplotype. This SNP was not present in 15 individuals of diverse racial and ethnic origins, in 186 Thai individuals, and in 133 of 134 Iranians living in Tehran. To our knowledge, this SNP has not been previously reported in the literature. To begin to study the functional significance of this SNP, gel mobility shift analysis was done with two 40 nt long oligomers, one with the wild type GATA sequence and the other with the mutated GAGA sequence, using uninduced mouse erythroleukemia (MEL) cell nuclear extracts. The mutant GAGA sequence results in a complete loss of GATA-1 binding. The region from nt −382 to −730 5′ to the Aγ-globin gene was reported to be related to Aγ-globin gene silencing (Stamatoyannopoulos et al, Mol Cell Biol 13:7636, 1993). The nt −567 T>G SNP is located within the comparable region of the Gγ-globin gene, which is highly homologous to the Aγ-globin gene. Among the 4 family members under study, no other SNPs are found in the same region. Taken together, these observations raise the possibility that the T>G SNP at nt −567 5′ to the Gγ-globin gene is associated with elevated Hb F, that might be caused by a novel mechanism, i.e., incomplete silencing of the Gγ-globin gene, resulting from the abolished GATA-1 binding. Additional clinical and functional studies will be needed to further document the effect of this SNP upon Gγ-globin gene expression and to ascertain that this SNP represents a HPFH mutation. Hematological Data of Family Father Mother Son Son Age 52 44 13 9 Hb 15.7 14.1 13.2 13.8 MCV 82 77 75 75 Hb A2 2.5 % 3.0 % 3.4 % 3.3 % Hb F 10.2 % 0.7 % 0.7 % 5.9 %

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Epigenetic Insights and Potential Modifiers as Therapeutic Targets in β–Thalassemia

Biomolecules ◽

10.3390/biom11050755 ◽

2021 ◽

Vol 11 (5) ◽

pp. 755

Author(s):

Nur Atikah Zakaria ◽

Md Asiful Islam ◽

Wan Zaidah Abdullah ◽

Rosnah Bahar ◽

Abdul Aziz Mohamed Yusoff ◽

...

Keyword(s):

Gene Expression ◽

Clinical Presentation ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Dna Hypomethylation ◽

Considerable Research ◽

Non Coding Rna ◽

Hbf Level ◽

Long Non Coding Rna ◽

Globin Gene Expression

Thalassemia, an inherited quantitative globin disorder, consists of two types, α– and β–thalassemia. β–thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the β–globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases β–globin expression. Down-regulation of γ–globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. β–thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the β–thalassemia patient’s response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single β–thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of β–thalassemia with α–thalassemia ameliorates the β–thalassemia clinical presentation. In conclusion, the management of β–thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

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EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

Blood ◽

10.1182/blood-2015-06-649087 ◽

2015 ◽

Vol 126 (16) ◽

pp. 1930-1939 ◽

Cited By ~ 48

Author(s):

Aline Renneville ◽

Peter Van Galen ◽

Matthew C. Canver ◽

Marie McConkey ◽

John M. Krill-Burger ◽

...

Keyword(s):

Gene Expression ◽

Gene Locus ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Erythroid Cells ◽

Adult Human ◽

Key Points ◽

Globin Gene Expression

Key Points EHMT1/2 inhibition increases human γ-globin and HbF expression, as well as mouse embryonic β-globin gene expression. EHMT1/2 inhibition decreases H3K9Me2 and increases H3K9Ac at the γ-globin gene locus in adult human erythroid cells.

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Effect of LCR 5′HS4 and 5′HS1 Deletions on β-Like Globin Gene Expression in β-Yac Transgenic Mice.

Blood ◽

10.1182/blood.v104.11.1209.1209 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1209-1209

Author(s):

Susanna Harju ◽

Halyna Fedosyuk ◽

Kenneth R. Peterson

Keyword(s):

Gene Expression ◽

Homologous Recombination ◽

Globin Gene ◽

Developmental Expression ◽

Mrna Levels ◽

Wild Type ◽

Rnase Protection ◽

Transgenic Lines ◽

Definitive Erythropoiesis ◽

Globin Gene Expression

Abstract A 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) was modified by homologous recombination to delete 2.9 Kb of cross-species conserved sequence similarity encompassing the LCR 5′HS4 (Δ5′HS4 β-YAC). Three transgenic mouse lines were established; each contained two intact copies of the β-globin locus as determined by long range restriction enzyme mapping (LRRM) and Southern blot hybridization analyses. Human ε-, γ- and β-globin, and mouse α- and ζ-globin mRNAs were measured by RNAse protection in hematopoietic tissues derived from staged embryos, fetuses and adult mice. No difference in the temporal pattern of globin transgene expression was observed between Δ5′HS4 β-YAC mice and wild-type β-YAC mice. In addition, quantitative per-copy human β-like globin mRNA levels were similar between Δ5′HS4 and wild-type β-YAC transgenic lines, although γ-globin gene expression was slightly increased in the fetal liver, while β-globin gene expression was slightly decreased in Δ5′HS4 β-YAC mice. These data are in contrast to data obtained from β-YAC mice containing a deletion of the 280 bp 5′HS4 core. In these mice, γ- and β-globin gene expression was significantly decreased during fetal definitive erythropoiesis and β-globin gene expression was decreased during adult definitive erythropoiesis. However, these data are consistent with the observation that deletion of the 5′HS core elements is more deleterious than large deletions of the 5′HSs. Together, the compiled deletion data supports the hypothesis that the LCR exists as a holocomplex in which the 5′HS cores form an active site and the flanking 5′HS regions constrain the holocomplex conformation. In this model, 5′HS core mutations are dominant negative, whereas larger deletions allow the LCR to fold into alternate holocomplex structures that function normally, albeit less efficiently. To complete the study on the contribution of the individual 5′HSs to LCR function, a 0.8 Kb 5′HS1 fragment was deleted in the 213 Kb β-YAC by homologous recombination. Two ΔHS1 β-YAC transgenic lines have been established; four additional founders were recently identified. Of the two lines, one contains two intact copies of the globin locus; the other contains four deleted copies, one of which extends from the LCR through just 5′ to the β-globin gene. For both lines, ε-globin gene expression was markedly reduced, approximately 5–10 fold, during primitive erythropoiesis. Developmental expression profiles and levels of the γ- and β-globin genes (in the line that contains loci including the β-globin gene) were unaffected by deletion of 5′HS1. Breeding of the remaining four founders to obtain F1 and F2 progeny for similar structure/function studies is in progress. Decreased expression of the β-globin gene is the first phenotype ascribed to a 5′HS1 mutation, suggesting that this HS does indeed have a role in LCR function.

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