Understanding α-globin gene regulation and implications for the treatment of β-thalassemia

Abstract Hereditary Persistence of Fetal Hemoglobin is a rare, heterogeneous and benign group of hereditary disorders with an abnormal switch from fetal to adult hemoglobin, resulting in high levels of Hb F in the adult stage. A total of six deletions related to HPFH have been described, associated with increased levels of both gamma chains. Three main hypotheses have been proposed to explain the relationship between these deletions and the non-suppression of gamma genes: the removal of competitive regions that interact with the LCR; the juxtaposition of enhancer elements; and the removal of silencers. Despite evidence to support these hypotheses, however, they are not conclusive. Recently, Xiang and cols (Abstract #1215, 2004 ASH Meeting; Blood, Volume 104, issue 11, November 16, 2004) developed a YAC construct with the whole beta-globin locus containing a deletion of approximately 83.5 Kb responsible for the HPFH-2. Unexpectedly, the gamma gene was completely silenced in the adult transgenic mice. These data suggest that other mechanisms could be involved in the increased levels of HbF in these conditions. The authors speculate that other regions upstream from the cluster may harbor this activity. We, herein, investigate the possible involvement of transcription factors, using the subtractive hybridization method to identify differentially expressed transcripts in reticulocytes from a normal subject and a HPFH-2 subject. We have identified 56 and 106 unique genes in the normal and HPFH-2 cDNA libraries, respectively. Some of these are transcription (zinc fingers and homeobox proteins) and chromatin remodeling (NAP and SWI like proteins) factors that could participate in globin gene regulation. These genes are located in cis or in trans to the deletion and their altered gene expression has been confirmed by Quantitative Real-time PCR in other two HPFH subjects. The data may present new clues about globin gene regulation, the increased expression of gamma gene in deletional HPFH and the dynamic organization of genes and chromosomes in cells.

Download Full-text

Transcriptional Regulation of γ-Globin Gene Expression by KLF4

Blood ◽

10.1182/blood.v116.21.645.645 ◽

2010 ◽

Vol 116 (21) ◽

pp. 645-645

Author(s):

Inderdeep S Kalra ◽

Md. M Alam ◽

Betty S Pace

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Sodium Butyrate ◽

Globin Gene ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Mrna Levels ◽

Klf4 Expression ◽

Globin Gene Regulation ◽

Globin Gene Expression

Abstract Abstract 645 Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in various gene promoters. The CACCC element is also critical for developmental regulation of the human γ-globin and β-globin genes; therefore studies to identify transcription factors that bind the CACCC element to alter gene expression are desirable. By microarray-based gene profiling, we identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased simultaneously with γ-globin silencing during in vitro erythroid maturation. Subsequent reverse transcription quantitative PCR (RT-qPCR) analysis confirmed KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively in erythroid progenitors from day 7 to day 28 with over 90% γ-globin gene silencing. The effects of known fetal hemoglobin inducers hemin (50μM) and sodium butyrate (2mM) on KLF factor expression was tested in K562 cells. Hemin and sodium butyrate increased KLF4 3-fold (p<0.05) and 13-fold (p<0.01) respectively while KLF12 was only induced by butyrate. Likewise, hemin treatment of KU812 leukemia cells, which actively express γ-globin and β-globin, produced a 7-fold increase in KLF4 (p<0.05) while KLF12 levels were not changed suggesting KLF4 may be directly involved in γ-globin gene regulation. To characterize its role further siRNA-mediated loss of function studies were performed in K562 cells. A 60% knockdown of KLF4 expression produced 40% attenuation of γ-globin transcription (p<0.05). To confirm this effect, rescue experiments were performed as follows: K562 cells were treated with 100nM siKLF4 alone or in combination with the pMT3-KLF4 expression vector (10 and 20μg) for 48 hrs. The 40% knockdown of γ-globin expression produced by siKLF4 was rescued to baseline levels after enforced pMT3-KLF4 expression (p<0.05). To establish whether KLF4 directly stimulates γ-globin promoter activity, we performed co-transfection of pMT3-KLF4 and the Gγ-promoter (-1500 to +36) cloned into the pGL4.17 Luc2/neo vector; a dose-dependent increase in luciferase activity (2- to 5-fold; p<0.001) was observed. Furthermore, enforced expression of pMT3-KLF4 augmented endogenous γ-globin expression 2-fold (p<0.01). Collectively, these studies suggest that KLF4 acts as a trans-activator of γ-globin gene transcription. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. At day 11 when γ-globin gene expression was maximal, siKLF4 treatment produced a 60% decrease in γ/β-globin mRNA levels (p<0.001). By contrast, enforced pMT3-KLF4 expression enhanced γ/β-globin 1.5-fold at day 11 and day 28 (after γ-globin silencing); HbF levels were induced 1.5-fold (p<0.05) which was demonstrated by enzyme-linked immunosorbent assay. To gain insights into the molecular mechanism of KLF4-mediated γ-globin regulation, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) were completed. Since CREB binding protein (CBP) is known to function as a co-activator for KLF1, 4 and 13, we also tested its role in γ-globin gene regulation. EMSA performed with K562 nuclear extract and a [γ-32P] labeled γ-CACC probe (-155 to -132 relative to the γ-globin cap site) produced three DNA-protein complexes; the addition of KLF4 or CBP antibody resulted in a marked decrease in intensity of all complexes suggesting these factors bind the γ-CACC element. ChIP assay demonstrated 10-fold and 20-fold chromatin enrichment with KLF4 and CBP antibody respectively (p<0.001) confirming in vivo binding at the γ-CACC region. Lastly, co-immunoprecipitation established protein-protein interaction between KLF4 and CBP in K562 cells. Future studies will investigate the role of CBP in KLF4-mediated γ-globin regulation which will provide molecular targets for fetal hemoglobin induction and treatment of sickle cell anemia and β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Joining the loops: β-Globin gene regulation

IUBMB Life ◽

10.1002/iub.129 ◽

2008 ◽

Vol 60 (12) ◽

pp. 824-833 ◽

Cited By ~ 62

Author(s):

Daan Noordermeer ◽

Wouter de Laat

Keyword(s):

Gene Regulation ◽

Globin Gene ◽

Globin Gene Regulation

Download Full-text

The role of methyl binding domain protein 2 (MBD2) in developmental globin gene regulation

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2006.10.054 ◽

2007 ◽

Vol 38 (2) ◽

pp. 142-143

Author(s):

Gordon D. Ginder ◽

Evan P. Kransdorf ◽

Jeremy W. Rupon ◽

Shou Zhen Wang ◽

Sheng Zu Zhu ◽

...

Keyword(s):

Gene Regulation ◽

Globin Gene ◽

Binding Domain ◽

Domain Protein ◽

Globin Gene Regulation

Download Full-text

Flanking HS-62.5 and 3′ HS1, and regions upstream of the LCR, are not required for β-globin transcription

Blood ◽

10.1182/blood-2006-04-014431 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1395-1401 ◽

Cited By ~ 45

Author(s):

M. A. Bender ◽

Rachel Byron ◽

Tobias Ragoczy ◽

Agnes Telling ◽

Michael Bulger ◽

...

Keyword(s):

Gene Regulation ◽

Globin Gene ◽

Gene Activation ◽

Regulatory Elements ◽

Chromatin Domain ◽

Open Domain ◽

Binding Factor ◽

Necessary And Sufficient ◽

Globin Gene Regulation ◽

Endogenous Locus

Abstract The locus control region (LCR) was thought to be necessary and sufficient for establishing and maintaining an open β-globin locus chromatin domain in the repressive environment of the developing erythrocyte. However, deletion of the LCR from the endogenous locus had no significant effect on chromatin structure and did not silence transcription. Thus, the cis-regulatory elements that confer the open domain remain unidentified. The conserved DNaseI hypersensitivity sites (HSs) HS-62.5 and 3′HS1 that flank the locus, and the region upstream of the LCR have been implicated in globin gene regulation. The flanking HSs bind CCCTC binding factor (CTCF) and are thought to interact with the LCR to form a “chromatin hub” involved in β-globin gene activation. Hispanic thalassemia, a deletion of the LCR and 27 kb upstream, leads to heterochromatinization and silencing of the locus. Thus, the region upstream of the LCR deleted in Hispanic thalassemia (upstream Hispanic region [UHR]) may be required for expression. To determine the importance of the UHR and flanking HSs for β-globin expression, we generated and analyzed mice with targeted deletions of these elements. We demonstrate deletion of these regions alone, and in combination, do not affect transcription, bringing into question current models for the regulation of the β-globin locus.

Download Full-text

Exploring epigenetic and microRNA approaches for γ-globin gene regulation

Experimental Biology and Medicine ◽

10.1177/15353702211028195 ◽

2021 ◽

pp. 153537022110281

Author(s):

Athena Starlard-Davenport ◽

Ashley Fitzgerald ◽

Betty S Pace

Keyword(s):

Gene Regulation ◽

Globin Gene ◽

Single Agent ◽

Unmet Need ◽

Fetal Hemoglobin ◽

Therapeutic Interventions ◽

Globin Genes ◽

Chromosome 11 ◽

Globin Gene Regulation

Therapeutic interventions aimed at inducing fetal hemoglobin and reducing the concentration of sickle hemoglobin is an effective approach to ameliorating acute and chronic complications of sickle cell disease, exemplified by the long-term use of hydroxyurea. However, there remains an unmet need for the development of additional safe and effective drugs for single agent or combination therapy for individuals with β-hemoglobinopathies. Regulation of the γ-globin to β-globin switch is achieved by chromatin remodeling at the HBB locus on chromosome 11 and interactions of major DNA binding proteins, such as KLF1 and BCL11A in the proximal promoters of the globin genes. Experimental evidence also supports a role of epigenetic modifications including DNA methylation, histone acetylation/methylation, and microRNA expression in γ-globin gene silencing during development. In this review, we will critically evaluate the role of epigenetic mechanisms in γ-globin gene regulation and discuss data generated in tissue culture, pre-clinical animal models, and clinical trials to support drug development to date. The question remains whether modulation of epigenetic pathways will produce sufficient efficacy and specificity for fetal hemoglobin induction and to what extent targeting these pathways form the basis of prospects for clinical therapy.

Download Full-text