Efficient depletion of DNMT3B has no effect on DNA methylation.

<p>(A) DNMT3B mRNA levels were determined after siRNA knockdown with DNMT3B siRNA #4 by quantitative RT-PCR analysis. Expression values are means of triplicates and were calculated relative to Lamin B1 expression. Error bars represent standard errors. ***P<0.001 relative to control siRNA, determined by student's t-test prior to normalization. (B) Comparison of Infinium 27 k methylation profiles of HCT-116 cells transfected with DNMT3B siRNA #4 or control siRNA. (C) Efficient depletion of DNMT3B protein in stably shRNA-transduced HCT-116 cells. DNMT3B protein levels were determined by immunoblot analysis using ß-actin as a loading control. The double band presumably reflects the expression of two (or more) DNMT3B isoforms. (D) Comparison of Infinium 27 k methylation profiles between HCT-116 cells stably transduced with lentiviruses containing DNMT3B shRNA #1 and control cells transfected with a control shRNA. (E) Infinium 450 k methylation analysis comparing methylation profiles of HCT-116 cells transduced with DNMT3B-shRNA #1 and control cells transduced with a non-targeting control shRNA. (F) Heatmap based on the Infinium 450 k data, showing hypermethylated CIMP markers in HCT-116 cells stably transduced with shRNA #1 or #2 targeting DNMT3B or with a non-targeting control shRNA. AVB, Average Beta value.</p

The hypermethylation phenotype is maintained in DNMT3B knockout cells.

<p>(A) Heatmap based on Infinium 27 k data illustrating the methylation levels of 67 CIMP marker genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (B) Boxplot illustrating methylation levels of 67 CIMP marker genes. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles, respectively. (C) Heatmap based on Infinium 27 k data illustrating the methylation levels of 543 hypermethylated PRC2 target genes in HCT-116 colon cancer cells and HCT-116 DNMT3B knockout cells (3BKO). (D) Boxplot illustrating methylation levels of PRC2 target genes. AVB, Average Beta value.</p

The hypermethylation phenotype is reversed in HCT-116 DNMT1; DNMT3B double knockout cells.

<p>(A, B) Boxplots illustrating the methylation levels of CIMP (A) and PRC2 (B) marker genes in parental HCT-116 cells (HCT-116), HCT-116 cells lacking DNMT3B (3BKO), HCT-116 cells with a hypomorphic DNMT1 allele (1KO), and HCT-116 DNMT1; DNMT3B double knockout cells (DKO) on Infinium 27 k methylation arrays. Lines in boxes denote medians, boxes the interquartile range, and whiskers the 2.5th and 97.5th percentiles. ***P<0.001 relative to parental HCT116 cells, determined by a Wilcoxon rank sum test. (C) Model for the role of DNMT3B during cancer development and progression. In normal cells, CpG islands are generally unmethylated and the corresponding genes are actively transcribed. During cancer development, certain regions become methylated by de novo methyltransferases such as DNMT3A and DNMT3B. Once the hypermethylation phenotype is established, the methylation pattern is maintained by the maintenance methyltransferase DNMT1 and becomes independent of DNMT3 enzymes. Only a strong reduction of overall methyltransferase activity causes the demethylation of hypermethylated cancer genes and inhibits the re-establishment by de novo methyltransferases.</p

DNMT3B expression in human cancer tissue and colon cancer cell lines.

<p>(A) Array Northern analysis of DNMT3B mRNA expression on Affymetrix HGU133Plus2.0 arrays in tumor (T) tissue samples compared to normal (N) tissues of the same organ. n indicates the number of analyzed samples for the specified tissue type. Data are shown as mean±SD; *P<0.05 relative to normal tissue (N), **P<0.01 relative to normal tissue (N), ***P<0.001 relative to normal tissue (N), determined by student's t-test. (B) Quantitative real-time PCR analysis of DNMT3B mRNA expression in colon cancer cell lines and normal cells (HMEC-T53, WI-38). Expression values are means of triplicates and were calculated relative to GAPDH expression. Error bars represent standard errors. (C) Immunoblot of DNMT3B protein expression in lysates from various colon cancer cell lines and normal cells (HMEC-T53, WI-38). ß-actin was used as a loading control.</p

Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents