by Thomas A. Paul, Juraj Bies, Donald Small, and Linda Wolff Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML by Thomas A. Paul, Juraj Bies, Donald Small, and Linda Wolff Blood Volume 115(15):3098-3108 April 15, 2010 ©2010 by American Society of Hematology

p15INK4b DNA methylation and expression in AML cell lines used in study. p15INK4b DNA methylation and expression in AML cell lines used in study. (A) Bisulfite sequencing of the p15INK4b exon 1 CpG island. Filled circles represent percentage of DNA methylation at CpG positions from 10 independently sequenced clones (● represents ≥ 50% methylcytosine; , 10%-49% methylcytosine). (B-C) Quantitative reverse-transcriptase (RT)–PCR analysis of p15INK4b, p14ARF, and p16INK4a expression in AML cell lines. Copies of cDNA per 100 ng of total RNA were calculated from a plasmid DNA standard curve and represent the averages of 3 independent experiments with error bars of SD. Thomas A. Paul et al. Blood 2010;115:3098-3108 ©2010 by American Society of Hematology

Distribution of H3K4me3, H3K27me3, and EZH2 at INK4b-ARF-INK4a differs in AML cell lines with DNA methylation. Distribution of H3K4me3, H3K27me3, and EZH2 at INK4b-ARF-INK4a differs in AML cell lines with DNA methylation. ChIP-on-chip analysis of enrichment of H3K4me3 (blue), H3K27me3 (red) on chromosome 9: 21800000 to 22450000 (A) and chromosome 9: 21953000 to 22003000 (B) in AML cell lines. Top axis represents H3K4me3; and bottom axis, H3K27me3. Enrichment of EZH2 (green; C) and H3K9me3 (orange; D) at the INK4b-ARF-INK4a region in AML cell lines. Normalized enrichment data (IP/input) are plotted on a log 2 scale. Thomas A. Paul et al. Blood 2010;115:3098-3108 ©2010 by American Society of Hematology

AML patient blasts with p15INK4b DNA methylation display reduced H3K4me3 enrichment at the promoter. AML patient blasts with p15INK4b DNA methylation display reduced H3K4me3 enrichment at the promoter. (A) Bisulfite sequencing of the p15INK4b exon 1 CpG island in AML patient samples. Filled circles represent the frequency of DNA methylation at CpG positions in sequenced clones (● represents ≥ 50% methylcytosine; , 10%-49% methylcytosine). (B) Quantitative RT-PCR analysis of p15INK4b expression in AML patient blasts. cDNA copies per 50 ng total RNA were calculated from a plasmid DNA standard curve and represent the averages of 3 independent experiments with error bars of SD. Quantitative PCR analysis (C) of H3K4me3 and H3K27me3 enrichment at the p15INK4b promoter. Data are averages of ΔΔCt compared with input DNA from 3 technical replicates with error bars of SD. ChIP-on-chip analysis of enrichment of H3K4me3 (blue) and H3K27me3 (red) on chromosome 9: 21953000 to 22003000 (D) and chromosome 9: 21990000 to 22003000 (E) in AML patient samples. Top axis represents H3K4me3; and bottom axis, H3K27me3. Normalized enrichment data (IP/input) are plotted on a log 2 scale. Thomas A. Paul et al. Blood 2010;115:3098-3108 ©2010 by American Society of Hematology

Reactivation of p15INK4b expression and loss of DNA methylation after treatment with DNMT or HDAC inhibitors. Reactivation of p15INK4b expression and loss of DNA methylation after treatment with DNMT or HDAC inhibitors. (A) Quantitative RT-PCR analysis of p15INK4b expression in KG-1, KG-1a, and AML-193 cells after reactivation with 5-aza-dC and/or TSA. (B) Quantitative RT-PCR for p15INK4b, p14ARF, and p16INK4a expression in KG-1cells after reactivation with 5-aza-dC and/or TSA. (A-B) *Increase in expression relative to control (P < .05, 1-way analysis of variance). (C) Quantitative RT-PCR for p15INK4b expression in AML patient blasts treated with 5-aza-dC or TSA. Quantitative RT-PCR data in both cell lines and clinical samples were normalized to control cells and calculated from ΔΔCt averages of 3 independent experiments with error bars of SD. *Significant increase in expression in cells treated with a combination of 5-aza-dC and TSA compared with cells treated with 5-aza-dC alone (P < .05, 1-way analysis of variance). (D) Bisulfite sequencing of the p15INK4b CpG island in individual clones from control and treated KG-1, AML-193, and AML8 blasts after treatment with 5-aza-dC and/or TSA. Filled circles represent percentage DNA methylation at individual CpG positions from 10 independently sequenced clones (● represents ≥ 50% methylcytosine; , 10%-49% methylcytosine). Thomas A. Paul et al. Blood 2010;115:3098-3108 ©2010 by American Society of Hematology

Reactivation of p15INK4b using DNMT and HDAC inhibitors is associated with an increase in H3K4me3 and maintenance of H3K27me3 in KG-1 and AML blasts. Reactivation of p15INK4b using DNMT and HDAC inhibitors is associated with an increase in H3K4me3 and maintenance of H3K27me3 in KG-1 and AML blasts. ChIP-quantitative PCR measured enrichment of H3K4me3 and H3K27me3 at the p15INK4b promoter after reactivation in KG-1 (A) and AML-193 cells (B). The enrichment of H3K4me3 (C) and H3K27me3 (D) measured at the p15INK4b promoter by quantitative PCR in clinical samples AML8, AML9, AML10, and AML11 patient blasts treated with DMSO (control), 5-aza-dC, or TSA. (C) *Significant increase in H3K4me3 enrichment compared with control treatment (P < .05, 1-way analysis of variance). Enrichment was calculated from ΔΔCt averages of 3 independent experiments with error bars of SD. (E) ChIP-on-chip for enrichment of H3K4me3 (blue) and H3K27me3 (red) at INK4b-ARF-INK4a in DMSO control and 5-aza-dC plus TSA-treated KG-1 and AML-193 cells. (F) ChIP-on-chip for enrichment of H3K4me3 (blue) and H3K27me3 (red) at INK4b-ARF-INK4a in AML8 patient blasts treated with control DMSO, 5-aza-dC, or TSA. Normalized enrichment data (IP/input) are plotted on a log 2 scale. Thomas A. Paul et al. Blood 2010;115:3098-3108 ©2010 by American Society of Hematology