Supplementary MaterialsS1 File: Table A, The list of chemical molecules used in the drug screen. folds after BIX-01294 treatment were listed. Table G, Different expressed genes of SMYD2 knockdown cell with or without rapamycin treatment. SMYD2 was knocked down by siRNA and the different expressed genes higher than 1.5 EGFR-IN-3 folds after rapamycin treatment were listed. Table H, The list of primers for real time RT-PCR used in the study. Table I, The list of siRNA sequences targeting SMYD2 in the study.(XLSX) pone.0116782.s001.xlsx (686K) GUID:?83C6E3C8-5E67-42BD-8B8C-60AF27850FE5 Data Availability StatementThe high throughput sequencing data have been uploaded to GEO database. And a URL was arranged as below: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ytkbseoivhcrpab&acc=GSE61255 Abstract Transcription regulation emerged to be one of the key mechanisms in regulating autophagy. Inhibitors of H3K9 methylation activates the expression of LC3B, as well as other autophagy-related genes, and promotes autophagy process. However, the detailed mechanisms of autophagy regulated by nuclear factors remain elusive. In this study, we performed a drug screen of SMYD2-/- cells and discovered that SMYD2 deficiency enhanced the cell death induced by BIX01294, an inhibitor EGFR-IN-3 of histone H3K9 methylation. BIX-01294 induces accumulation of LC3 II and autophagy-related cell death, but not caspase-dependent apoptosis. We profiled the global gene expression pattern after treatment with BIX-01294, in comparison with rapamycin. BIX-01294 selectively activates the downstream genes of p53 signaling, such as p21 and DOR, but not EGFR-IN-3 PUMA, a typical p53 target gene inducing apoptosis. BIX-01294 also induces other autophagy-related Rabbit Polyclonal to HGS genes, such as ATG4A and ATG9A. SMYD2 is a methyltransferase for p53 EGFR-IN-3 and regulates its transcription activity. Its deficiency enhances the BIX-01294-induced autophagy-related cell death through transcriptionally promoting the expression of p53 target genes. Taken together, our data suggest BIX-01294 induces autophagy-related cell death and selectively activates p53 target genes, which is repressed by SMYD2 methyltransferase. Introduction Protein methylation on histones is initially well demonstrated in transcription regulation and chromatin structure [1, 2]. Later, methylation on non-histone proteins is also proved to be one of the key steps in regulating protein functions [3]. The protein methyltransferase family of SET and MYND domain containing proteins is of important functions in tumorigenesis and development processes [4]. These proteins contain an atypical SET domain, which is split into two parts by one MYND domain [4]. SMYD proteins exert their function by methylating proteins on lysines, among which SMYD2 (SET and MYND domain containing 2) is the mostly studied. SMYD2 is initially identified as a methyltransferase for histone H3K36 and H3K4 [5, 6]. Till now, the SMYD2 target sites on chromatin are still not well demonstrated, however, since it mainly localizes in the cytoplasma, SMYD2 has important functions on non-histone proteins. Multiple proteins were identified as the substrates of SMYD2, such as p53 (tumor protein p53), Rb (retinoblastoma 1), HSP90 (heat shock protein 90kDa), PARP1 (poly (ADP-ribose) polymerase 1) and ESR1 (estrogen receptor 1) [7C11]. SMYD2 methylates p53 at Lys370 and represses p53 transcription activity [7]. Since p53 and Rb are among the most well-known tumor suppressor genes, SMYD2 is considered a potential oncogene. Several studies reported that SMYD2 is overexpressed in the tumor cells lines and patients tissues of some cancer types, including esophageal squamous cell carcinoma and acute lymphoblastic leukemia, which suggests SMYD2 as a potential drug target in these cancers [9, 12, 13]. The tissues with most abundant SMYD2 expression include heart, brain and muscle [14]. Surprising, SMYD2 deficiency in cardiomyocyte is usually dispensable for heart development [14]. Recently, one report proved SMYD2 represses p53 activity and cardiomyocyte apoptosis induced by cobalt chloride, which suggested SMYD2 as a regulatory protein in stress response [15]. In order to.