unc-0638 and Anemia--Sickle-Cell

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies.

Topics: Adult; Anemia, Sickle Cell; beta-Thalassemia; Cell Differentiation; DNA-Binding Proteins; Enzyme Inhibitors; Epigenesis, Genetic; Erythroid Precursor Cells; Erythropoiesis; Fetal Hemoglobin; gamma-Globins; Histocompatibility Antigens; Histone-Lysine N-Methyltransferase; Humans; In Vitro Techniques; LIM Domain Proteins; Locus Control Region; Models, Biological; Promoter Regions, Genetic; Quinazolines; Transcription Factors

Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic β-globin genes Hbb-εy and Hbb-βh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.

Topics: Anemia, Sickle Cell; Animals; Cell Line, Tumor; Epigenesis, Genetic; Erythroblasts; Erythroid Cells; Female; Fetal Hemoglobin; Histocompatibility Antigens; Histone-Lysine N-Methyltransferase; Humans; Male; Mice; Quinazolines