Chen Chih-Cheng, Chiu Chia-Chen, Lee Kuan-Der, Hsu Chia-Chen, Chen Hong-Chi, Huang Tim H-M, Hsiao Shu-Huei, Leu Yu-Wei
Department of Hematology and Oncology, Chang Gung Memorial Hospital, Chiayi, Chang Gung University College of Medicine, Taoyuan, Taiwan; Chang Gung Institute of Technology, Taoyuan, Taiwan.
Department of Hematology and Oncology, Chang Gung Memorial Hospital, Chiayi, Chang Gung University College of Medicine, Taoyuan, Taiwan; Chang Gung Institute of Technology, Taoyuan, Taiwan; Human Epigenomics Center, Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, National Chung Cheng University, Chia-Yi, Taiwan.
Biochem Biophys Res Commun. 2017 Dec 16;494(3-4):470-476. doi: 10.1016/j.bbrc.2017.10.108. Epub 2017 Oct 21.
Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2-STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were <3% and 0.4%, respectively, as identified through pSTAT3 and EZH2 ChIP-seq. Furthermore, the methylation changes in the direct target loci (FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo.
在Janus激酶（JAK2）617密码子处缬氨酸（V）到苯丙氨酸（F）的负向转换（V617F）是骨髓增殖性肿瘤（MPN）患者的主要驱动突变。JAK2V617F被证明足以导致细胞转化；然而，独立突变可能会影响随后的表观基因组修饰。为了在无干扰的情况下评估JAK2V617F诱导的下游表观基因组变化，我们分析了Ba/F3细胞中异位表达的JAK2V617F的表观基因组变化。针对磷酸化信号转导和转录激活因子3（pSTAT3）和zeste同源物2增强子（EZH2）的抗体用于染色质免疫沉淀测序（ChIP-seq），以检测JAK2-STAT3信号通路中的下游表观基因组靶点。为了在体内确认JAK2V617F诱导的表观遗传变化，通过甲基化特异性聚合酶链反应检测了MPN患者靶位点的DNA甲基化变化，并与对照组的变化进行聚类分析。我们发现，Ba/F3细胞中异位表达的JAK2V617F降低了结合特异性；它与顺式调控元件相关，并在pSTAT3下游和EZH2相关靶点中识别DNA基序。通过pSTAT3和EZH2 ChIP-seq鉴定，对照组和JAK2V617F之间的重叠靶位点分别<3%和0.4%。此外，在分析的MPN样本中，直接靶位点（FOXH1、HOXC9和SRF）的甲基化变化与对照位点（L1TD1）和其他突变基因（HMGA2和Lin28A）独立聚类。因此，JAK2V617F影响pSTAT3和EZH2中的靶标结合。在表观遗传调节因子无突变的情况下，JAK2V617F可诱导下游表观基因组修饰。因此，JAK2下游靶点的表观遗传变化在体内可能是可追踪的。
