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

Abstract

The genes of the vertebrate ␤-globin locus undergo a switch in expression during erythroid development whereby embryonic͞ 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 ␥-globin gene. The ␥-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human ␤-globin locus as a yeast artificial chromosome (␤YAC) transgene. To elucidate the mechanism through which DNA methylation represses the ␥-globin gene in adult erythroid cells, ␤YAC͞MBD2؊/؊ mice were generated by breeding ␤YAC mice with MBD2؊͞؊ mice. Adult ␤YAC͞MBD2؊/؊ mice continue to express the ␥-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 ␥-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 embryonic͞fetal erythroid development. DNA methylation levels are modestly decreased in MBD2؊͞؊ mice. MBD2 does not bind to the ␥-globin promoter region to maintain ␥-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of ␥-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain ␥-globin suppression in adult erythroid cells.DNA methylation ͉ epigenetics ͉ transcription

Identification of a PRMT5-dependent repressor complex linked to silencing of human fetal globin gene expression

“…23 MBD2 has recently been implicated in silencing of the human ␥-genes in transgenic mice, although in this setting it was not localized to the ␥-promoter. 34 MBD2 has also been linked to silencing of the chicken embryonic genes, where it binds in a repressor complex containing NuRD components. 35 The identification of both MBD2 and MBD3 in the ␥-globin gene PRMT5-dependent repressor complex is in contrast to the PRMT5-containing complex that assembles on the P14 ARF and P16 INK4a CpG islands, which contains only MBD2.…”

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

“…Furthermore, studies carried out in baboons, which have a fetal-to-adult globin switch that is similar to the one seen in humans, show a correlation between gene expression and globin promoter (promoter methylation and globin gene expression are inversely related) [46]. Finally, in transgenic mice carrying a human β-globin YAC, targeted deletion of the methyl-CpG binding protein MBD2 gene delays γ-globin developmental silencing [47]. Recently, the pattern of γ-and β-globin promoter methylation was explored in primary human fetal liver and adult bone marrow erythroid cells.…”

Persistent Fetal γ-Globin Expression in Adult Transgenic Mice following Deletion of Two Silencer Elements Located 3′ to the Human Aγ-Globin Gene

“…All the above studies strongly indicate that apart from the elements located within the Aγ-to δ-globin intergenic region, hemoglobin switching is a consequence of a complex interplay between developmental stage-specific transcription factors which interact both within the promoters of the various genes and the 5′ LCR, as well as of specific modifications of the physical location of a gene within the locus (47,(57)(58)(59). Thus, sequencespecific transcription factors (activators and/or repressors), such as the recently identified developmental stage-specific repressor BCL11A (10), can regulate ε-and γ-globin activation and repression through nearby cis elements in a gene autono mous manner, independently of the amplifying effects of the LCR.…”