Expression of the leukemic prognostic marker CD7 is linked to epigenetic modifications in chronic myeloid leukemia

Abstract

Background: Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells.

Results: To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression.

Conclusion: These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.

Figure 1

Expression of CD7 and ELA2 is associated with DNA hypomethylation in leukemic cell lines. Bisulfite sequencing and CoBRA for A) ELA2, on THP-1 (positive) and RPM1 (negative); and B) CD7 on THP-1 (negative) and ALL-SIL (positive). Cartoons show gene organization, with transcriptional start sites as bent arrow, UTR as narrow bars and translated regions as wide bars. Down arrows show location of enzyme sites used for CoBRA. Vertical bars show location of CpG dinucleotides. Dotted lines show location of region amplified after ChIP. Open and filled circles indicate unmethylated and methylated CpGs respectively. Bottom panels show CoBRA for each gene. Enzymes used are indicated as follows; U = uncut, H = HinfI, R = Rsa, T = TaqIα and M = MboI. Position of the uncut band is indicated by an arrow.

Figure 2

Expression of CD7 and ELA2 is associated with histone acetylation in leukemic cell lines. ChIP was performed on THP-1 (CD7^-ELA2⁺) and ALL-SIL (CD7⁺ELA2^-) cell lines with anti-acetyl-H3K9 and anti-pan-acetyl-H4 antibodies. Black bars indicate NFM (negative control); grey bars, CD7; white bars, ELA2 and striped bars, HPRT (positive control).

Figure 3

Absolute quantification of transcripts of A) CD7, B) ELA2 and C) PRTN3 in normal and CML human samples. Normal samples are all from bone marrow, CML 1-10 are from peripheral blood except for CML11 and 12 which are from bone marrow. Cells and cDNA were prepared as described in materials and methods. Median of transcript levels is indicated.

Figure 4

DNA methylation analysis of CD7 in normal and CML samples. CD7 is expressed highly in CML1 and intermediately in CML10 but CML4 and CML6 have close to normal levels of CD7 expression. Open and black fill circles indicate unmethylated and methylated CpG respectively.

Figure 5

Significantly less CD7 promoter methylation in CD7 expressing cells from CML1 and CML10. CD34⁺ cells of patient sample CML1 and CML10 were sorted into CD7⁺ and CD7^- fractions and DNA methylation analysis was performed by sodium bisulfite sequencing. Open and black fill circles indicate unmethylated and methylated CpG respectively.

Figure 6

DNA methylation analysis of ELA2 in normal and CML samples. Open and black fill circles indicate unmethylated and methylated CpG respectively. Grey fill circles indicated CpG dinucleotides whose DNA methylation could not be determined. Two fragments of ~300 bp and ~400 bp were amplified and sequenced. The percentage of methylated CpGs is calculated independently for each fragment.