Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN

Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the microenvironment on globin gene expression. Adult progenitors express high levels of c-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, c-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in c-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and c-globin promoter DNA methylation. We conclude that c-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction

Hydroxymethylcytosine and demethylation of the <i>γ-globin</i> gene promoter during erythroid differentiation

<div><p>The mechanism responsible for developmental stage-specific regulation of γ<i>-globin</i> gene expression involves DNA methylation. Previous results have shown that the γ<i>-globin</i> promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ<i>-globin</i> gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ<i>-globin</i> gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ<i>-globin</i> promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ<i>-globin</i> expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ<i>-globin</i> promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ<i>-globin</i> promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ<i>-globin</i> promoter.</p></div

Roles of DNA methylation and development in modulation of γ-globin expression in AFT024 co-cultures. A.

<p>Bisulfite sequence analysis of DNA methylation of the γ-globin 5′ promoter region in erythroid progenitors grown in methylcellulose media, liquid media, or in co-cultures containing the AFT024 stromal cell line. Five CpG sites numbered relative to the transcription start site were analyzed. Each row represents the sequence analysis of a single amplicon cloned within the pCR4 vector. Unmethylated sites (green), methylated site (red), polymorophic sites where no CpG is present (yellow). <b>B.</b> Effect of decitabine on globin chain synthesis (γ/γ+β) during erythroid differentiation of baboon BM progenitors in AFT024 co-cultures. Decitabine (5×10⁻⁷ M) was added to cultures on d7. Globin chain synthesis was analyzed on d11 and d14. <b>C.</b> Globin chain synthesis (γ/γ+β) during erythroid differentiation of human cord blood progenitors in AFT024 co-cultures.</p

Effect of stromal cell line co-cultures on γ-globin expression during erythroid differentiation of CD34+ bone marrow progenitors.

<p><b>A.</b> Comparison of globin chain synthesis on varying days in baboon BM-derived erythroid progenitors grown in liquid media and in AFT024 stromal cell line co-cultures. <b>B.</b> Globin chain synthesis in baboon BM-derived erythroid progenitors grown in methylcellulose media (MC), liquid media (LM), and co-cultures containing the AFT024, U26B1, or OP9 stromal cell lines. <b>C.</b> Comparison of globin mRNA expression (γ/γ+β) in baboon BM-derived erythroid progenitors grown in methylcellulose media, liquid media, or AFT024 stromal cell line co-cultures. <b>D.</b> Globin chain synthesis in CD34+ baboon BM subpopulations differing in CD36 expression.</p

Effect of AFT024 and U26B1 cell lines on γ-globin expression requires cell-cell contact.

<p>Analysis of γ-globin chain synthesis (γ/γ+β) in baboon BM-derived CD34+ cells cultured in methylcellulose (MC), liquid media (LM), and AFT024 and U26 B1 co-cultures, transwell (TW) cultures and in LM containing conditioned media (CM) from the AFT024 and U26B1 cell lines. The single asterisk denotes statistical significance (p<.01) relative to AFT co-cultures. The double asterisk denotes statistical significance (p<.01) relative to U26B1 co-cultures.</p