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

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Biochemistry, Biophysics and Molecular Biology

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Introduction

Materials and Methods

Comparative analysis of the genomic loci of tnni1b

Analysis of the putative TFBSs

Construction of the TF overexpression, enhancer activity detection and mutation analysis constructs

Zebrafish maintenance, microinjection, and transgenic line generation

Cell culture, transfection, and luciferase reporter assay

Protein extraction and electrophoretic mobility shift assay (EMSA)

Fluorescent image processing and analysis

Statistical analysis

Results

Identification of a 183 bp ECR located 84 kb upstream of tnni1b

The core region, tnni1b-ECR183-d2, is only 87 bp

Tnni1b-ECR183-d2 drives the specific expression of GFP near the atrioventricular junction

Analysis of the enhancer activity of tnni1b-ECR183-d2 by a luciferase assay

Tnni1b-ECR183-d2 has positive correlations with NKX2.5 or JUN

Functional analysis of human enhancers

Discussion

Conclusions

Supplemental Information

Information of embryo numbers and GFP expression from transient injections of tnni1b-ECR183, tnni1b-ECR183-d1, tnni1b-ECR183-d2, tnni1b-ECR183-d3,tnni1b-ECR183-h179 and tnni1b-ECR183-h84

DOI: 10.7717/peerj.10289/supp-1

Putative transcription factors binding sites (TFBSs) of tnni1b-ECR183 in PROMO and JASPAR

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Identification of zebrafish enhancer activity and analysis of putative TFBSs by luciferase reporter assay

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Identification of human enhancer activity and analysis of putative TFBSs by luciferase reporter assay

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Lateral views of zebrafish embryos with GFP expression outside of the heart after injection with tol2 mRNA and tnni1b-ECR183

Scale bars = 100 µm.

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Binding reactions between tnni1b-ECR183-d2 and NKX2.5 (lanes 1–4), ETS1 (lanes 5–8) and JUN (lanes 9–12) based on the EMSA

Lanes 13-15 show the binding reactions of the control EBNA system. Specific binding reactions are shown in lanes 1, 5, 9 and 13; competition reactions are shown in lanes 2, 6, 10 and 14; mutation reactions are shown in lanes 3, 7 and 11; and negative reactions are shown in lanes 4, 8, 12 and 15.

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Primers of oligos for target DNA synthesis in EMSA

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Information on 16 ECRs in the 219 kb zebrafish genomic region

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Heart-specific GFP expression of embryos injected with tnni1b-ECR183-d2 at 24 hpf

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Heart-specific GFP expression of embryos injected with tnni1b-ECR183-d2 at 48 hpf

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Heart-specific GFP expression of embryos injected with tnni1b-ECR183-d2 at 72 hpf

DOI: 10.7717/peerj.10289/supp-11

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Yawen Zhang and Feng Wang performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Fang Wu performed the experiments, prepared figures and/or tables, and approved the final draft.

Youhua Wang, Xu Wang, Yonghao Gui and Qiang Li conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The Institutional Animal Care and Use Committee of Children’s Hospital of Fudan University approved this research (approval number: EK(2018)73).

Data Availability

The following information was supplied regarding data availability:

The raw data are available in the Supplementary Files.

Funding

This work was supported by grants from the National Natural Science Foundations of China (grant No. 81771632) and the National Key Research and Development Program (grant No. 2016YFC1000500) to Qiang Li; by the National Natural Science Foundations of China (grant No. 81470442, 81741081, 81873481 and 81170147), 973 Program (grant No. 2013CB945401) to Yonghao Gui; and by the Shanghai Key Laboratory of Birth Defects (grant No. 13DZ2260600). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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