利用ChIP-seq/SILAC技术筛选KLF15的靶向基因SUMO1

Screening of the target gene SUMO1 of KLF15 with the ChIP-seq/SILAC techniques

目的利用ChIP-seq和SILAC-LC/MS技术寻找Krupple样因子(KLF15)的靶向基因及其结合位点。方法 (1)利用染色质免疫沉淀技术(ChIP)特异性富集并纯化人原代肾系膜细胞(HRMC)中可与KLF15结合的DNA片段,通过高通量测序技术(Hi Seq)检测和分析,得到直接与KLF15结合的基因;(2)利用稳定同位素标记细胞培养技术(SILAC),重链和轻链同位素标记的培养HRMC,分别对重链实验组(HK)转染KLF15质粒,轻链对照组(LC)转染空载体对照,收集蛋白进行液质谱(LC/MS)检测,得到过表达KLF15后的差异蛋白;(3)对ChIP-seq及SILAC结果进行GO和Pathway分析,同时将二者进行交集筛选出KLF15的靶向基因,而后利用http://meme.edi.edu.au/网站获得靶基因的Modifs并对候选基因小泛素样修饰因子1(SUMO1)进行ChIP-PCR及双荧光素酶验证。以SPSS 17.0统计软件进行统计学分析。结果 ChIP-seq获取了2 478个KLF15直接调控的可能靶向基因,SILAC-LC/MS检测到KLF15过表达后有1 357个差异蛋白,综合ChIP和SILAC结果,我们分析得到52个共同差异表达的基因和(或)蛋白及其3个modifs,其中5个蛋白参与细胞增殖相关进程;结合GO和Pathway分析结果,最终筛选出SUMO1作为KLF15调控系膜细胞增殖的靶向基因。Motif分析发现SUMO1启动子区存在KLF15的结合序列;利用ChIP-PCR和双荧光素酶报告基因验证KLF15转录因子可以直接结合SUMO1的启动子区并发挥作用。结论 KLF15通过结合SUMO1的启动子区,调节SUMO1的表达。

Objective This article aimed to determine the target genes and binding sites of Krupplelike factor 15（ KLF15） with the ChIP-seq and SILAC-LC/MS techniques. Methods（ 1） The chromatin immunoprecipitation（ ChIP） method was used to specifically isolate and purify the KLF15-binding DNA fragments from the human primary renal mesangial cells（ HRMC）. And the KLF15-binding genes were obtained with the high-throughput sequencing（ Hi Seq） technique;（ 2） By means of the cell culture plus stable isotope labeling with amino acids（ SILAC） technique as well as the heavy chain or light chain isotope labeled HRMCs,the heavy chain test group（ HK） was transfected with the KLF15 plasmids,while the light chain control group（ LC） was transfected with the vector control plasmids. Then the proteins were collected for detection with the liquid chromatography/mass spectrometry（ LC/MS） method so that the differential proteins were obtained after KLF15 overexpression;（ 3） The ChIP-seq and SILAC results were analyzed with the GO/Pathway method and the target genes of KLF15 were screened and selected. The modifs of the target gene were got from the http：//meme. edi. edu. au/website,and the candidate gene small ubiquitin-like modifier 1（ SUMO1） was verified with the ChIP-PCR and dual luciferase methods. Statistical analysis was performed with the SPSS17. 0 software. Results ChIP-seq method detected 2 478 potential target genes that were directly regulated by KLF15,and SILAC-LC/MS detected 1357 differential proteins after KLF15 overexpression. By combining the results of ChIP and SILAC detections,52 common genes/proteins together with their 3 modifs emerged,of which 5 proteins involved in cell proliferation-related process. By combining the results of both the GO and Pathway analyses,SUMO1 was finally screened out as a target gene of KLF15 for regulating the renal mesangial cell proliferation. Motif analysis located a KLF15-binding sequence in the SUMO1 promoter region. ChIP-PCR and dual-luciferase reporter genes verified that transcription factor KLF15 was able to directly bind to the promoter region of SUMO1 to come into play. Conclusion KLF15 regulated the expression of SUMO1 by binding to the promoter region of SUMO1.