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

Abstract

Objective This article aimed to determine the target genes and binding sites of Krupple-like 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 (HiSeq) 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. Key words: Krupple-like factor 15; Renal mesangial cells; ChIP-seq; SILAC; SUMO1