Epigenetic dysregulation of the erythropoietic transcription factor KLF1 and the β-like globin locus in juvenile myelomonocytic leukemia

Abstract

Increased levels of fetal hemoglobin (HbF) are a hallmark of more than half of the children diagnosed with juvenile myelomonocytic leukemia (JMML). Elevated HbF levels in JMML are associated with DNA hypermethylation of distinct gene promoter regions in leukemic cells. Since the regulation of globin gene transcription is known to be under epigenetic control, we set out to study the relation of DNA methylation patterns at β-/γ-globin promoters, mRNA and protein expression of globins, and epigenetic modifications of genes encoding the globin-regulatory transcription factors BCL11A and KLF1 in nucleated erythropoietic precursor cells of patients with JMML. We describe several altered epigenetic components resulting in disordered globin synthesis in JMML. We identify a cis-regulatory upstream KLF1 enhancer sequence as highly sensitive to DNA methylation and frequently hypermethylated in JMML. The data indicate that the dysregulation of β-like globin genes is a genuine attribute of the leukemic cell clone in JMML and involves mechanisms not taking part in the normal fetal-to-adult hemoglobin switch.

Keywords: Epigenetic regulation; KLF1; fetal hemoglobin; juvenile myelomonocytic leukemia.

Figure 1.

β-like globin expression in glycophorin A-positive erythroid precursor cells. (A) Isolation of GPEP cells and other nucleated cells from cryopreserved spleen cells of JMML patients, in vitro-differentiated blood cells of healthy adults and cord blood cells of healthy newborns. (B) β-like globin mRNA level [γ/(γ+β) mRNA quotient] measured by RT-qPCR in GPEP cells of adults, newborns, and JMML patients. (C) High pressure liquid chromatography to determine hemoglobin variants in GPEP cells of 3 patients with JMML. HbF0/HbA0/HbA2: unmodified, HbF1: glycosylated; for the calculation of %HbF as HbF/(HbF+HbA) the amount of unmodified and glycosylated HbF was considered. For each patient, the corresponding globin mRNA quotient is indicated above the chromatogram. (D) Correlation between globin mRNA quotient and %HbF in GPEP cells of 3 patients with JMML. (E) HBB transcript levels relative to the GAPDH reference gene in GPEP cells of adults, JMML patients classified as normal HbF, and JMML patients classified as elevated HbF. (F) HBG transcript levels relative to GAPDH in GPEP cells of adults, JMML patients classified as normal HbF, and JMML patients classified as elevated HbF. Due to high sequence homology, the assay did not discriminate between HBG1 and HBG2. (G) Total β-like globin transcription relative to GAPDH in GPEP cells of adults, JMML patients classified as normal HbF, and JMML patients classified as elevated HbF. Mann-Whitney test: NS, not significant; **P ≤ 0.01; ***P ≤ 0.001. MNC, mononuclear cells; HEMA, human erythroid massive amplification; CB, cord blood.

Figure 2.

DNA methylation of the β-like globin gene promoters. (A) Target region (gray) in the γ-globin (HBG) promoter with CpG dinucleotide sites that were analyzed for methylation by mass spectrometry. Due to high sequence homology, the assay did not discriminate between HBG1 and HBG2. (B) γ-globin promoter CpG methylation (mean of 6 CpG sites) in GPEP cells of adults, newborns, JMML patients classified as normal HbF, and JMML patients classified as elevated HbF. (C) Correlation between globin expression [γ/(γ+β) mRNA quotient] and γ-globin promoter methylation in GPEP cells of patients with JMML. r, Pearson coefficient. (D) γ-globin promoter CpG methylation (mean of 6 CpG sites) in non-GPEP cell populations of adults, newborns, and patients with JMML. (E) Target region (gray) in the β-globin (HBB) promoter with CpG dinucleotide sites that were analyzed for methylation by bisulfite sequencing. (F) β-globin promoter CpG methylation in GPEP cells of 6 JMML patients assessed by bisulfite sequencing. Lines represent individual alleles; filled circles, methylated cytosines; open circles, unmethylated cytosines. The average level of methylation across CpG sites and alleles is indicated at the top for each patient. Mann-Whitney test: **p ≤ 0.01; ***p ≤ 0.001.

Figure 3.

DNA methylation analysis of the KLF1 gene. (A) Regulation of β-like globin expression in erythropoietic cells by transcription factors BCL11A and KLF1. LCR, locus control region. (B) Target regions (gray) in the KLF1 gene locus that were analyzed for methylation by mass spectrometry. (C) CpG methylation of the KLF1 enhancer region (mean of 5 CpG sites) in GPEP cells of adults, newborns, and patients with JMML. (D) KLF1 enhancer methylation in non-GPEP cell populations of adults, newborns, and patients with JMML. (E) KLF1 enhancer methylation in the erythroleukemia cell line K562 and the non-erythroid myeloid leukemia cell line HL60. Mann-Whitney test: NS, not significant; **P ≤ 0.01.

Figure 4.

Hypermethylation inhibits the activity of the KLF1 enhancer. (A) Analysis of KLF1 enhancer activity by a luciferase reporter assay in K562 cells. Luciferase activity was normalized to the activity of the pCpGL-CMV/EF1 Firefly control vector. Normalized activity of the construct containing unmethylated KLF1 enhancer CpG sites and the construct containing methylated KLF1 enhancer CpG sites are shown relative to the normalized activity of the reporter construct not containing the KLF1 enhancer sequence (empty vector). Error bars represent standard deviation of 3 transfection experiments. T test: ****P ≤ 0.0001. (B) Same experiment in non-haematopoietic HEK293T cells, confirming specificity of KLF1 enhancer function to erythropoietic cells.

Figure 5.

Expression analysis of KLF1 and its regulatory target CD44. (A) KLF1 transcript levels relative to the GAPDH reference gene measured by reverse transcriptase quantitative polymerase chain reaction in GPEP cells of adults, newborns, and patients with JMML. Cells of a healthy adult were used as calibrator (relative log2 expression = 0). Mann-Whitney test: *P ≤ 0.05; **P ≤ 0.01. (B) Correlation between KLF1 enhancer methylation and KLF1 mRNA expression in GPEP cells of patients with JMML. r, Pearson coefficient. (C) Correlation between KLF1 and HBB mRNA expression in GPEP cells of patients with JMML. r, Pearson coefficient. (D) Surface expression of CD44 on glycophorin A-negative mononuclear (non-GPEP) cells (left panel) or GPEP cells (right panel) of 3 JMML patients and a healthy control measured by flow cytometry. APC, allophycocyanin. (E) KLF1 enhancer methylation (left panel) and KLF1 mRNA expression relative to GAPDH (right panel) in GPEP cells of 3 JMML patients analyzed in.Fig. 2D Cells of a healthy control were used as calibrator (dashed line).