Gillinder Kevin R, Tuckey Hugh, Bell Charles C, Magor Graham W, Huang Stephen, Ilsley Melissa D, Perkins Andrew C
Cancer Genomics Group, Mater Research Institute - University of Queensland, Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia.
Faculty of Medicine and Biomedical Sciences, University of Queensland, St. Lucia, Brisbane, Queensland, Australia.
PLoS One. 2017 Jul 21;12(7):e0180922. doi: 10.1371/journal.pone.0180922. eCollection 2017.
Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.
促红细胞生成素（EPO）通过二聚体促红细胞生成素受体发挥作用，以刺激红系祖细胞的增殖、存活、分化和去核。我们采用了两种互补的方法来寻找小鼠红系细胞中EPO依赖性的pSTAT5靶基因：对新转录的（4sU标记的）RNA进行RNA测序，以及在EPO刺激30分钟后对pSTAT5进行染色质免疫沉淀测序（ChIP-seq）。我们发现了302个pSTAT5占据位点：其中约15%位于启动子中，其余位于内含子增强子或基因间区域，有些距离最近的转录起始位点（TSS）超过100kb。大多数pSTAT5峰包含一个中心回文GAS元件，即TTCYXRGAA。在pSTAT5结合峰的附近，GATA基序和CACCC盒基序有显著富集，并且在许多位点存在GATA1和/或KLF1的共占据。使用4sU-RNA测序，我们确定了EPO诱导的转录组，并使用动态帽分析基因表达（CAGE）数据和定量逆转录聚合酶链反应（qRT-PCR）验证了差异表达基因。我们鉴定出了已知的直接pSTAT5靶基因，如Bcl2l1、Pim1和Cish，以及许多可能参与驱动红系细胞分化的新靶基因，包括那些参与mRNA剪接（Rbm25）、表观遗传调控（Suv420h2）和EpoR周转（Clint1/EpsinR）的基因。这些新的EpoR-JAK2-pSTAT5靶基因中的一些可作为生物标志物，用于监测真性红细胞增多症的疾病活动，以及监测对JAK抑制剂的反应。
