人NPTX1相关H3K27ac富集的非保守性增强子鉴定  

Identification of human NPTX1 associated non-conserved enhancers from H3K27ac-enrichments

作　　者：陈旭东 张琦[1,2,3,4] 张颖蓝 林佳[1,2,3,4] 王凤 桂怡婷 刘婷[1,2,3,4] 李强 CHEN Xu-dong;ZHANG Qi;ZHANG Ying-lan;LIN Jia;WANG Feng;GUI Yi-ting;LIU Ting;LI Qiang(Institute of Pediatrics,National Children's Medical Center/Children’s Hospital,Fudan University,Shanghai 201102,China;Translational Medical Center for Child Development and Disease,Shanghai 201102,China;Shanghai Key Laboratory of Birth Defect Prevention and Control,Shanghai 201102,China;National Health Commission Key Laboratory of Neonatal Diseases(Fudan University),Shanghai 201101,China)

机构地区：[1]国家儿童医学中心/复旦大学附属儿科医院儿科研究所,上海201102 [2]复旦大学儿童发育与疾病转化医学研究中心,上海201102 [3]上海市出生缺陷防治重点实验室,上海201102 [4]国家卫生健康委员会新生儿疾病重点实验室(复旦大学),上海201102

出　　处：《复旦学报（医学版）》2023年第4期487-493,501,共8页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(81771632,81271509);上海市自然科学基金(21ZR1410100)。

摘　　要：目的探索和鉴定人NPTX1相关H3K27ac富集的非保守性的增强子,完善NPTX1相关调控区的注释,为NPTX1相关疾病及非编码区突变的研究提供相应的理论基础。方法从CistromeDB数据库下载和分析涉及7种组织的39个H3K27ac ChIP-seq数据,选择NPTX1两侧100 kb范围内的峰。通过ECR浏览器分析各片段在人、小鼠和斑马鱼之间的序列保守性。将相应DNA片段插入双荧光素酶验证载体中,在人神经源细胞系SH-SY5Y和人肾源细胞系293T行双荧光素酶实验,以检测增强子活性。结果分析获得5个H3K27ac富集片段,各片段在人、小鼠和斑马鱼之间DNA序列不保守。NPTX1-E1-P在SH-SY5Y中双荧光素酶活性比值明显高于阴性对照组,但在293T细胞中其增强子活性下降约40%。结论NPTX1附近的H3K27ac富集片段中,NPTX1-E1-P在人神经细胞系SH-SY5Y中具有增强子活性,且在物种间序列保守性低。相对于人神经源细胞系SH-SY5Y,其在人肾源细胞系293T中增强子活性下降。Objective To explore and identify H3K27ac enriched non-conserved enhancers near human NPTX1,improve the annotation of NPTX1-related regulatory regions,and provide a corresponding theoretical basis for the study of NPTX1-related diseases and mutations in non-coding regions.Methods A total of 39 H3K27ac ChIP-seq data involving 7 tissues were downloaded from the CistromeDB database and analyzed.Peaks in the range of 100 kb on both sides of NPTX1 were selected.Fragments’sequence conservation among human,mouse and zebrafish was analyzed by ECR browser.The fragments were inserted into a dual luciferase validation vector,and dual luciferase assays were performed in human neural-derived cell line SH-SY5Y and human kidney-derived cell line 293T to detect enhancers’activity.Results We obtained 5 H3K27ac-enriched fragments.The DNA sequences of each fragment were not conserved among human,mouse and zebrafish.The ratio of dual luciferase activity of NPTX1-E1-P was significantly higher than that of the negative control in SH-SY5Y,but it was about 40%lower in 293T cells.Conclusion Among the H3K27ac-enriched fragments near NPTX1,NPTX1-E1-P has enhancer activity in human neurogenic cell line SH-SY5Y,and low sequence conservation among species.Compared with the human neurogenic cell line SH-SY5Y,its enhancer activity is decreased in human kidney-derived cell line 293T.

关 键 词：NPTX1 增强子 染色质免疫共沉淀测序(ChIP-seq) H3K27ac 

分 类 号：R34[医药卫生—基础医学]