Tnni1b - ECR183 - d2，一种斑马鱼的87碱基对心脏增强子。

Tnni1b-ECR183-d2, an 87 bp cardiac enhancer of zebrafish.

作者信息

Zhang Yawen, Wang Feng, Wu Fang, Wang Youhua, Wang Xu, Gui Yonghao, Li Qiang

机构信息

Translational Medical Center for Development and Disease, Shanghai Key Laboratory of Birth Defect, Institute of Pediatrics, Children's Hospital of Fudan University, Shanghai, China.

Department of Cardiology, Children's Hospital of Fudan University, Shanghai, China.

出版信息

PeerJ. 2020 Nov 4;8:e10289. doi: 10.7717/peerj.10289. eCollection 2020.

DOI:10.7717/peerj.10289

PMID:33194440

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7648457/

Abstract

BACKGROUND

Several heart malformations are associated with mutations in the regulatory regions of cardiac genes. is important for the formation of the atrioventricular canal in zebrafish hearts; however, the regulation of is poorly understand. We aimed to identify a small but functional enhancer that is distal to .

METHODS

Evolutionary Conserved Region (ECR) Browser was used to analyze the 219 kb zebrafish and human genomes covering the gene as well as the 100 kb regions upstream and downstream of . Putative transcription factor binding sites (TFBSs) were analyzed using JASPAR and PROMO, and the enhancer activity was identified using zebrafish embryos and the luciferase reporter assay. A correlation analysis between the enhancer and transcription factors (TFs) was performed via TF overexpression and TFBS mutation experiments and the electrophoretic mobility shift assay (EMSA). To analyze the conservation between zebrafish and human enhancers, human DNA fragments were functionally verified. Images were captured and analyzed by fluorescence microscopy or confocal microscopy.

RESULTS

Combined with comparative analysis and functional validation, we identified a 183 bp ECR (termed tnni1b-ECR183) that was located approximately 84 kb upstream of that had the heart-specific enhancer activity in zebrafish. TFBS analysis and the enhancer activity detection assay data showed that the 87 bp core region (termed tnni1b-ECR183-d2) was capable of driving specific GFP expression near the atrioventricular junction and increased luciferase expression in HEK293 and HL1 cell lines. The GFP pattern in zebrafish embryos was similar to the expression profiles of . A correlation analysis showed that the enhancer activity of tnni1b-ECR183-d2 was increased when NKX2.5 ( = 0.0006) or JUN ( < 0.0001) was overexpressed and was decreased when the TFBSs of NKX2.5 ( < 0.0001) or JUN ( = 0.0018) were mutated. In addition, DNA-protein interactions were not observed between these TFs and tnni1b-ECR183-d2 in the EMSA experiment. The conservation analysis showed that tnni1b-ECR183-h179 (aligned from tnni1b-ECR183) drove GFP expression in the heart and skeletal muscles and increased the luciferase expression after NKX2.5 ( < 0.0001), JUN ( < 0.0001) or ETS1 ( < 0.0001) was overexpressed. Interestingly, the truncated fragment tnni1b-ECR183-h84 mainly drove GFP expression in the skeletal muscles of zebrafish and the enhancer activity decreased when NKX2.5 ( = 0.0028), ETS1 ( = 0.0001) or GATA4 ( < 0.0001) was overexpressed.

CONCLUSIONS

An 87 bp cardiac-specific enhancer located 84 kb upstream of in zebrafish was positively correlated with NKX2.5 or JUN. The zebrafish and human enhancers in this study target different tissues. The GFP expression mediated by tnni1b-ECR183-d2 is a valuable tool for marking the domain around the atrioventricular junction.

摘要

背景

几种心脏畸形与心脏基因调控区域的突变有关。在斑马鱼心脏中，对于房室管的形成很重要；然而，其调控机制尚不清楚。我们旨在鉴定一个位于远端的小的功能性增强子。

方法

使用进化保守区域（ECR）浏览器分析覆盖基因的219 kb斑马鱼和人类基因组以及该基因上下游100 kb的区域。使用JASPAR和PROMO分析推定的转录因子结合位点（TFBSs），并使用斑马鱼胚胎和荧光素酶报告基因检测鉴定增强子活性。通过TF过表达、TFBS突变实验和电泳迁移率变动分析（EMSA）进行增强子与转录因子（TFs）之间的相关性分析。为了分析斑马鱼和人类增强子之间的保守性，对人类DNA片段进行了功能验证。通过荧光显微镜或共聚焦显微镜捕获并分析图像。

结果

结合比较分析和功能验证，我们鉴定出一个183 bp的ECR（称为tnni1b - ECR183），其位于斑马鱼心脏特异性增强子活性的上游约84 kb处。TFBS分析和增强子活性检测实验数据表明，87 bp的核心区域（称为tnni1b - ECR183 - d2）能够驱动房室交界处附近的特异性GFP表达，并增加HEK293和HL1细胞系中的荧光素酶表达。斑马鱼胚胎中的GFP模式与的表达谱相似。相关性分析表明，当NKX2.5（= 0.0006）或JUN（< 0.0001）过表达时，tnni1b - ECR183 - d2的增强子活性增加，而当NKX2.5（< 0.0001）或JUN（= 0.0018）的TFBSs发生突变时，增强子活性降低。此外，在EMSA实验中未观察到这些TF与tnni1b - ECR183 - d2之间的DNA - 蛋白质相互作用。保守性分析表明，tnni1b - ECR183 - h179（与tnni1b - ECR183比对）在心脏和骨骼肌中驱动GFP表达，并在NKX2.5（< 0.0001）、JUN（< 0.0001）或ETS1（< 0.0001）过表达后增加荧光素酶表达。有趣的是，截短片段tnni1b - ECR183 - h84主要在斑马鱼的骨骼肌中驱动GFP表达，并且当NKX2.5（= 0.0028）、ETS1（= 0.0001）或GATA4（< 0.0001）过表达时，增强子活性降低。