Dataset Information

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

ABSTRACT: Background:Several heart malformations are associated with mutations in the regulatory regions of cardiac genes. Troponin I type 1b (tnni1b) is important for the formation of the atrioventricular canal in zebrafish hearts; however, the regulation of tnni1b is poorly understand. We aimed to identify a small but functional enhancer that is distal to tnni1b. Methods:Evolutionary Conserved Region (ECR) Browser was used to analyze the 219 kb zebrafish and human genomes covering the tnni1b gene as well as the 100 kb regions upstream and downstream of tnni1b. 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 tnni1b 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 tnni1b. A correlation analysis showed that the enhancer activity of tnni1b-ECR183-d2 was increased when NKX2.5 (p = 0.0006) or JUN (p < 0.0001) was overexpressed and was decreased when the TFBSs of NKX2.5 (p < 0.0001) or JUN (p = 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 (p < 0.0001), JUN (p < 0.0001) or ETS1 (p < 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 (p = 0.0028), ETS1 (p = 0.0001) or GATA4 (p < 0.0001) was overexpressed. Conclusions:An 87 bp cardiac-specific enhancer located 84 kb upstream of tnni1b 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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC7648457 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Zhang Yawen Y Wang Feng F Wu Fang F Wang Youhua Y Wang Xu X Gui Yonghao Y Li Qiang Q

PeerJ 20201104

<h4>Background</h4>Several heart malformations are associated with mutations in the regulatory regions of cardiac genes. <i>Troponin I type 1b (tnni1b)</i> is important for the formation of the atrioventricular canal in zebrafish hearts; however, the regulation of <i>tnni1b</i> is poorly understand. We aimed to identify a small but functional enhancer that is distal to <i>tnni1b</i>.<h4>Methods</h4>Evolutionary Conserved Region (ECR) Browser was used to analyze the 219 kb zebrafish and human gen ...[more]

PMID: 33194440

Similar Datasets

Project description:BACKGROUND: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. DESCRIPTION: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. CONCLUSION: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure