Chicken α‐globin switching depends on autonomous silencing of the embryonic π globin gene by epigenetics mechanisms

Abstract

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

“…Finally, we also examined In addition, this model postulates that inactivation of π gene transcription in erythroid cells of definite lineage is mediated by formation of a repressive chromatin domain containing the π gene. 42 Our data do not support this model. First, we failed to demonstrate that the downstream enhancer or the MRE (which the authors cited above do not consider in their model) interacts with the π gene promoter in erythroid cells of primitive lineage.…”

“…40,41 CpG methylation of the π gene promoter and neighboring regions in erythroblasts of definite lineage has been reported by several authors. [40][41][42] Methylated CpG islands recruit the transcriptional repressor MeCP2, which represses promoter activity directly 43 or through the recruitment of histone deacetylases, which remove active chromatin marks from the histone tails. 43,44 This model is in agreement with the observations of the present study.…”

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes

“…7a). Chicken red blood cells (RBC) CTCF ChIP experiments were performed as previously described 44,45 . We evaluated the CTCF-ChIP quality with several positive and negative PCR controls (Supplementary Figure 7b).…”

An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression

“…CpG, represents a CpG-island located ∼4-kb upstream of the embryonic π globin gene which represents the first CTCF-binding site identified in vivo , that corresponds to the promoter of the antisense transcript C16orf35 (47). Vertical arrows indicate the position of DNase I hypersensitive sites and the 3′-side enhancer (21). ( B ) Flow cytometry histograms (FACS) showing the GFP fluorescence level of five representative and independent stably transfected cell lines containing the control vector without insulators at days 0, 30 and 100 of continuous cell culture.…”