Methyl binding domain protein 2 mediates γ-globin gene silencing in adult human βYAC transgenic mice

Massey Cancer Center and Departments of Internal Medicine, Microbiology and Immunology, and Human Genetics, Virginia Commonwealth University, Richmond, VA 23298-0037, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2006; 103(17):6617-22. DOI: 10.1073/pnas.0509322103
Source: PubMed


The genes of the vertebrate beta-globin locus undergo a switch in expression during erythroid development whereby embryonic/fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal gamma-globin gene. The gamma-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human beta-globin locus as a yeast artificial chromosome (betaYAC) transgene. To elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2-/- mice were generated by breeding betaYAC mice with MBD2-/- mice. Adult betaYAC/MBD2-/- mice continue to express the gamma-globin gene at a level commensurate with 5-azacytidine treatment, 10- to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of gamma-globin expression is consistently higher in MBD2-/- mice in 14.5- and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic/fetal erythroid development. DNA methylation levels are modestly decreased in MBD2-/- mice. MBD2 does not bind to the gamma-globin promoter region to maintain gamma-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of gamma-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells.

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Available from: Joyce A Lloyd, Oct 01, 2015
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    • "Histone modification or repressor complex (e.g. NURD complex) reconstitution which might trigger histone modification changes may contribute to the γ-globin gene reactivation [21-23]. Alternatively, we cannot exclude the possibility that different erythroid specific transcription factors play roles upon γ-globin induction by Adox treatment [24,25]. "
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    ABSTRACT: Background Pharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells. Methods Expression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP). Results Adox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5). Conclusions The ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.
    Journal of Translational Medicine 01/2013; 11(1). DOI:10.1186/1479-5876-11-14 · 3.93 Impact Factor
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    • "Therefore we proceeded to analyze GFP transcript induction by qPCR, and normalized the relative fold enrichment by setting respective controls to 1. In agreement with previous work [10], [12], [25]–[27] all treatments induced GFP expression, i.e. γ-globin reactivation, and AZA was the most potent activator (Figure S2B). "
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    ABSTRACT: The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics.
    PLoS ONE 12/2012; 7(12):e51272. DOI:10.1371/journal.pone.0051272 · 3.23 Impact Factor
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    • "It is generally accepted that two mechanisms account for transcriptional repression via DNA methylation. In addition to directly inhibiting the binding of transcription factors, such as AP-2, c-Myc, E2F, and NF-κB to their binding sites within promoter [30], methylation of CpG islands in non-cis-element containing regions, for example extrons or introns, could present a significant obstacle to the processive transcription complexes by altering chromatin structure, thus inhibiting transcription elongation [31]. Since there are no transcription factor binding sites in the second region of the KIAA1199 CpG island, we assume that prevention of transcription elongation occurs in non-KIAA1199 expressing cells resulting in little to no expression of KIAA1199. "
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    ABSTRACT: Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.
    PLoS ONE 09/2012; 7(9):e44661. DOI:10.1371/journal.pone.0044661 · 3.23 Impact Factor
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