切换至 "中华医学电子期刊资源库"

中华肾病研究电子杂志 ›› 2018, Vol. 07 ›› Issue (02) : 74 -81. doi: 10.3877/cma.j.issn.2095-3216.2018.02.006

所属专题: 文献

论著

利用ChIP-seq/SILAC技术筛选KLF15的靶向基因SUMO1
李鸥1, 徐华2, 马倩1, 王旭1, 尹忠1, 吴玲玲1, 谢院生1, 蔡广研1, 陈香美1, 洪权1,()   
  1. 1. 100853 北京,解放军总医院肾脏病科、解放军肾脏病研究所、肾脏疾病国家重点实验室、国家慢性肾病临床医学研究中心
    2. 100850 军事医学研究院毒物药物研究所
  • 收稿日期:2018-03-05 出版日期:2018-04-28
  • 通信作者: 洪权
  • 基金资助:
    国家自然科学基金(81470949,81330019)

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

Ou Li1, Hua Xu2, Qian Ma1, Xu Wang1, Zhong Yin1, Lingling Wu1, Yuansheng Xie1, Guangyan Cai1, Xiangmei Chen1, Quan Hong1,()   

  1. 1. Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing 100853
    2. Institute of Toxicology and Pharmacology, Academy of Military Medical Research, Beijing 100850; China
  • Received:2018-03-05 Published:2018-04-28
  • Corresponding author: Quan Hong
  • About author:
    Corresponding author: Hong Quan, Email:
引用本文:

李鸥, 徐华, 马倩, 王旭, 尹忠, 吴玲玲, 谢院生, 蔡广研, 陈香美, 洪权. 利用ChIP-seq/SILAC技术筛选KLF15的靶向基因SUMO1[J]. 中华肾病研究电子杂志, 2018, 07(02): 74-81.

Ou Li, Hua Xu, Qian Ma, Xu Wang, Zhong Yin, Lingling Wu, Yuansheng Xie, Guangyan Cai, Xiangmei Chen, Quan Hong. Screening of the target gene SUMO1 of KLF15 with the ChIP-seq/SILAC techniques[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2018, 07(02): 74-81.

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(HiSeq)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 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.

图1 生物进程、细胞组成、分子功能基因的GO分析
图2 细胞进程、细胞周期进程基因的功能分析
图3 分子功能中生长基因、细胞生长基因功能分析
图4 SILAC差异蛋白的GO富集分析(评分前30)
图5 SILAC差异蛋白的Pathway分析(评分前30)
图6 综合ChIP和SILAC数据筛选结果
表1 综合ChIP和SILAC筛选出的52个KLF15靶基因数据
Refseq编号 基因名 基因描述
NM_002940 ABCE1 ATP结合盒蛋白,亚家族E(OABP),成员1[ATP-binding cassette, sub-family E (OABP), member 1]
NM_002197 ACO1 可溶性,顺乌头酸酶1(aconitase 1, soluble)
NM_005100 AKAP12 AMP蛋白激活激酶结合蛋白12[A kinase (PRKA) anchor protein 12]
NM_000477 ALB 白蛋白(albumin)
NM_001627 ALCAM 白细胞活化黏附分子(activated leukocyte cell adhesion molecule)
NM_002860 ALDH18A1 醛脱氢酶18家族,成员A1(aldehyde dehydrogenase 18 family, member A1)
NM_000700 ANXA1 膜联蛋白A1(annexin A1)
NM_004039 ANXA2 膜联蛋白A2(annexin A2)
NM_000701 ATP1A1 ATP酶,Na/K转运α1多肽(ATPase, Na/K transporting, alpha 1 polypeptide)
NM_004281 BAG3 BCL结合抗凋亡基因3(BCL2-associated athanogene 3)
NM_001221 CAMK2D 钙/钙调蛋白依赖蛋白激酶2δ(calcium/calmodulin-dependent protein kinase II delta)
NM_001344 DAD1 抗细胞死亡因子1(defender against cell death 1)
NM_001539 DNAJA1 DnaJ(Hsp40)同源物,亚族A,成员1[DnaJ (Hsp40) homolog, subfamily A, member 1]
NM_012100 DNPEP 天冬氨酰氨基肽酶(aspartyl aminopeptidase)
NM_001415 EIF2S3 真核翻译起始因子2,亚组3γ(eukaryotic translation initiation factor 2, subunit 3 gamma, 52kDa)
NM_001568 EIF3E 真核转录起始因子3,亚组E(eukaryotic translation initiation factor 3, subunit E)
NM_021814 ELOVL5 超长链脂肪酸延长酶5(elongase for very long fatty acid 5)
NM_014673 EMC2 内质网膜蛋白复合体2(ER membrane protein complex subunit 2)
NM_006832 FERMT2 丝裂原诱导蛋白2(fermitin family member 2)
NM_000583 GC 维生素D结合蛋白[group-specific component (vitamin D binding protein)]
NM_000518 HBB 血红蛋白β(hemoglobin, beta)
NM_000521 HEXB 己糖苷酶β[hexosaminidase B (beta polypeptide)]
NM_002138 HNRNPD 非均一核核糖蛋白D(heterogeneous nuclear ribonucleoprotein D)
NM_001553 IGFBP7 胰岛素样生长因子结合蛋白7(insulin-like growth factor binding protein 7)
NM_005578 LPP 脂肪瘤中LIM结构域(LIM domain containing preferred translocation partner in lipoma)
NM_000240 MAOA 单胺氧化酶A(monoamine oxidase A)
NM_005909 MAP1B 微管相关蛋白1B(microtubule-associated protein 1B)
NM_013283 MAT2B 甲硫氨酸腺苷基转移酶2β(methionine adenosyltransferase II, beta)
NM_002395 ME1 NADP(+)依赖型苹果酸酶1[malic enzyme 1, NADP(+)-dependent, cytosolic]
NM_002415 MIF 巨噬细胞迁移抑制因子(糖基化抑制因子)[macrophage migration inhibitory factor (glycosylation-inhibiting factor)]
NM_005956 MTHFD1 亚甲基四氢叶酸脱氢酶1[methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1]
NM_002526 NT5E 5′-核苷酸酶(CD73)[5′-nucleotidase, ecto (CD73)]
NM_013341 OLA1 Spo0B相关GTP结合蛋白基因样ATP酶1(Obg-like ATPase 1)
NM_002583 PAWR 蛋白激酶C凋亡调节因子(PRKC, apoptosis, WT1, regulator)
NM_013374 PDCD6IP 程序性细胞死亡作用蛋白6(programmed cell death 6 interacting protein)
NM_004911 PDIA4 蛋白质二硫键异构酶A4(protein disulfide isomerase family A, member 4)
NM_003681 PDXK 吡哆醛激酶[pyridoxal (pyridoxine, vitamin B6) kinase]
NM_021105 PLSCR1 磷脂脱氢酶1(phospholipid scramblase 1)
NM_002734 PRKAR1A cAMP依赖型蛋白激酶调节因子1α(protein kinase, cAMP-dependent, regulatory, type I, alpha)
NM_012293 PXDN 果蝇过氧化蛋白同源物[peroxidasin homolog (Drosophila)]
NM_012414 RAB3GAP2 RAB3 GTP酶激活蛋白2[RAB3 GTPase activating protein subunit 2 (non-catalytic)]
NM_002901 RCN1 内质网钙结合蛋白1(reticulocalbin 1, EF-hand calcium binding domain)
NM_001032 RPS29 核糖体蛋白S29(ribosomal protein S29)
NM_016106 SCFD1 四肽类超家族蛋白1(sec1 family domain containing 1)
NM_014766 SCRN1 分离蛋白1(secernin 1)
NM_021199 SQRDL 硫化物醌还原酶[sulfide quinone reductase-like (yeast)]
NM_003352 SUMO1 小类泛素华修饰分子(small ubiquitin-like modifier 1)
NM_004607 TBCA 微管折叠因子A(tubulin folding cofactor A)
NM_006759 UGP2 尿苷二磷酸葡萄糖焦磷酸化酶2(UDP-glucose pyrophosphorylase 2)
NM_016226 VPS29 空泡蛋白S29[vacuolar protein sorting 29 homolog (S. cerevisiae)]
NM_006826 YWHAQ 酪氨酸酶3-单加氧酶/色氨酸5-单加氧酶激活蛋白(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta)
NM_080660 ZC3HAV1L 锌指CCCH型抗病毒分子1L(zinc finger CCCH-type, antiviral 1-like)
图7 52个差异基因的Motif分析
图8 KLF15结合SUMO1启动子的靶区
图9 ChIP-PCR的初步验证情况
图10 双荧光素酶(Luc、Rluc)基因检测报告
[1]
Xu X, Ning Y, Shang W, et al. Analysis of 4931 renal biopsy data in central China from 1994 to 2014 [J]. Ren Fail, 2016, 38(7):1021-1030.
[2]
Couser WG, Johnson RJ. Mechanisms of progressive renal disease in glomerulonephritis [J]. Am J Kidney Dis, 1994, 23(2):193-198.
[3]
Kashgarian M, Sterzel RB. The pathobiology of the mesangium [J]. Kidney Int, 1992, 41(3):524-529.
[4]
Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases [J]. Nat Rev Mol Cell Biol, 2013, 14(8):518-528.
[5]
Ahn JD, Morishita R, Kaneda Y, et al. Transcription factor decoy for AP-1 reduces mesangial cell proliferation and extracellular matrix production in vitro and in vivo [J]. Gene Ther, 2004, 11(11):916-923.
[6]
Tomita N, Kim JY, Gibbons GH, et al. Gene therapy with an E2F transcription factor decoy inhibits cell cycle progression in rat anti-Thy 1 glomerulonephritis [J]. Int J Mol Med, 2004, 13(5):629-636.
[7]
Kang JH, Chae YM, Park KK, et al. Suppression of mesangial cell proliferation and extracellular matrix production in streptozotocin-induced diabetic rats by Sp1 decoy oligodeoxynucleotide in vitro and in vivo [J]. J Cell Biochem, 2008, 103(2):663-674.
[8]
Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins [J]. Mol Cell Biol, 1993, 13(5):2776-2786.
[9]
McConnell BB, Yang VW. Mammalian Kruppel-like factors in health and diseases [J]. Physiol Rev, 2010, 90(4):1337-1381.
[10]
Gao X, Wu G, Gu X, et al. Kruppel-like factor 15 modulates renal interstitial fibrosis by ERK/MAPK and JNK/MAPK pathways regulation [J]. Kidney Blood Press Res, 2013, 37(6):631-640.
[11]
Gu X, Xu D, Fu L, et al. KLF 15 works as an early anti-fibrotic transcriptional regulator in Ang II-induced renal fibrosis via down-regulation of CTGF expression [J]. Kidney Blood Press Res, 2017, 42(6):999-1012.
[12]
Mallipattu SK, Guo Y, Revelo MP, et al. Kruppel-like factor 15 mediates glucocorticoid-induced restoration of podocyte differentiation markers [J]. J Am Soc Nephrol, 2017, 28(1):166-184.
[13]
Hong Q, Li C, Xie Y, et al. Kruppel-like factor-15 inhibits the proliferation of mesangial cells [J]. Cell Physiol Biochem, 2012, 29(5-6):893-904.
[14]
Zhang J, Zhong HB, Lin Y, et al. KLF15 suppresses cell proliferation and extracellular matrix expression in mesangial cells under high glucose [J]. Int J Clin Exp Med, 2015, 8(11):20330-20336.
[15]
Gao L, Qiu H, Liu J, et al. KLF15 promotes the proliferation and metastasis of lung adenocarcinoma cells and has potential as a cancer prognostic marker [J]. Oncotarget, 2017, 8(66):109952-109961.
[16]
Sun C, Ma P, Wang Y, et al. KLF15 inhibits cell proliferation in gastric cancer cells via up-regulating CDKN1A/p21 and CDKN1C/p57 expression [J]. Dig Dis Sci, 2017, 62(6):1518-1526.
[17]
Wang K, Ren Y, Liu Y, et al. miR-4262 promotes proliferation and invasion of human breast cancer cells through directly targeting KLF6 and KLF15 [J]. Oncol Res, 2017, 25(2):277-283.
[18]
Elnitski L, Jin VX, Farnham PJ, et al. Locating mammalian transcription factor binding sites: a survey of computational and experimental techniques [J]. Genome Res, 2006, 16(12):1455-1464.
[19]
Klein RH, Hu W, Kashgari G, et al. Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation [J]. J Biol Chem, 2017, 292(46):18937-18950.
[20]
Ying M, Tilghman J, Wei Y, et al. Kruppel-like factor-9 (KLF9) inhibits glioblastoma stemness through global transcription repression and integrin alpha6 inhibition [J]. J Biol Chem, 2014, 289(47):32742-32756.
[21]
Park PJ. ChIP-seq: advantages and challenges of a maturing technology [J]. Nat Rev Genet, 2009, 10(10):669-680.
No related articles found!
阅读次数
全文


摘要