Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation by Rodwell Mabaera, Christine A. Richardson, Kristin Johnson, Mei Hsu, Steven Fiering, and Christopher H. Lowrey Blood Volume 110(4):1343-1352 August 15, 2007 ©2007 by American Society of Hematology

Characterization of purified fetal and adult nucleated erythroid cells. Characterization of purified fetal and adult nucleated erythroid cells. (A) Wright-Giemsa–stained fetal liver and adult marrow cells before and after purification by ficol density gradient and anti-glycophorin-A–conjugated magnetic beads. (B) Percentage of glycophorin-A (CD235a)–positive cells before and after purification. (C) Relative levels of globin gene mRNA in purified cells from fetal and adult erythroid cells determined by quantitative RT-PCR. Results are normalized to the geometric mean of β-actin and G3PD mRNAs as described in “Materials and methods; Quantitative RT-PCR and Hb HPLC.” Values are plus or minus 1 standard deviation. (D) Hb HPLC patterns from purified fetal liver and adult erythroid cells. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Methylation of γ-globin promoter sequences. Methylation of γ-globin promoter sequences. (A) Diagram of the human γ-globin promoter regions showing the locations of binding sites for transcription factors and CpG dinucleotides. Adapted from Stamatoyannopoulos and Grosveld.26 (B) Representative methylation data for the 2 γ-globin promoters in fetal and adult erythroid cells. □ indicates unmethylated; ■, methylated; and ▩, indeterminate sequencing. (C) Summary methylation data from multiple independent tissue samples. Error bars indicate plus or minus 1 standard deviation. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Methylation of β-globin promoter sequences. Methylation of β-globin promoter sequences. (A) Diagram of the human β-globin promoter region showing the locations of binding sites for transcription factors and CpG dinucleotides. Adapted from Stamatoyannopoulos and Grosveld.26 (B) Representative methylation data for the β-globin promoter in fetal and adult erythroid cells. □ indicates unmethylated; ■, methylated; and ▩, indeterminate sequencing. (C) Summary methylation data from multiple independent tissue samples. Error bars indicate plus or minus 1 standard deviation. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Methylation of γ- and β-globin promoter sequences in fetal hepatocytes and adult CD34+ peripheral blood hematopoietic cells. Methylation of γ- and β-globin promoter sequences in fetal hepatocytes and adult CD34+ peripheral blood hematopoietic cells. (A) Methylation of the 2 γ-globin promoters and the β-globin promoter in purified fetal hepatocytes. Data from 2 independent samples are shown. (B) Methylation of the γ- and β-globin promoters in CD34+ peripheral blood cells. □ indicates unmethylated; ■, methylated; and ▩, indeterminate sequencing. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Histone H3 acetylation at the γ- and β-globin promoters in purified fetal and adult erythroid cells determined by ChIP assay. Histone H3 acetylation at the γ- and β-globin promoters in purified fetal and adult erythroid cells determined by ChIP assay. (A) Relative promoter H3 acetylation in nucleated fetal erythroid cells. (B) Relative promoter H3 acetylation in nucleated adult erythroid cells. Results are expressed as fold enrichment using anti-AcH3 antibody compared with nonspecific IgG. Error bars indicate plus or minus 1 standard deviation. β-Globin LCR HS2 and HS3 core regions serve as positive controls. The necdin gene promoter, which is not expressed in erythroid cells, serves a negative control. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

In vitro erythroid differentiation from purified human CD34+ peripheral blood hematopoietic cells. In vitro erythroid differentiation from purified human CD34+ peripheral blood hematopoietic cells. (A) Wright-Giemsa–stained cytospin samples during in vitro differentiation. (B) Flow cytometric characterization of cells during differentiation. Scatter plots show CD45/CD34 staining and CD71 (transferrin)/CD235 (glycophorin A) at start and completion of differentiation. Graph shows the changing patterns of CD34 and CD235a cell surface expression during in vitro differentiation. (C) Expansion of cells during in vitro differentiation. (D) Relative levels of γ- and β-globin mRNA during in vitro differentiation as determined by quantitative RT-PCR. Values are normalized to the geometric mean of β-actin and G3PD mRNAs as described in “Materials and methods; Quantitative RT-PCR and Hb HPLC.” (E) Patterns of steady-state γ- and β-globin mRNA during differentiation. Values are expressed as the proportion of maximal expression to highlight the time course of expression. (F) Hb HPLC performed on cellular extracts at the end of the culture period. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Changing patterns of γ-globin promoter methylation during in vitro differentiation. Changing patterns of γ-globin promoter methylation during in vitro differentiation. (A) Percentage methylation of specific CpGs in the γ-globin promoters during in vitro differentiation. (B) Comparison of percentage methylation of the 3 CpGs upstream of the transcriptional start site to that of the 3 CpGs downstream of the start site. (C) Relative mRNA levels of DNMT genes during in vitro differentiation. Error bars indicate plus or minus 1 standard deviation. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology

Comparison of γ-globin mRNA, DNMT1 mRNA, and upstream γ-globin promoter methylation levels during in vitro differentiation. Comparison of γ-globin mRNA, DNMT1 mRNA, and upstream γ-globin promoter methylation levels during in vitro differentiation. The maximum levels of γ-globin and DNMT1 mRNA were set to 100%. DNA methylation is the percentage methylation of the upstream (−162, −53, and −50) CpGs and is not normalized. Rodwell Mabaera et al. Blood 2007;110:1343-1352 ©2007 by American Society of Hematology