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

PeerJ
Yawen ZhangQiang Li

Abstract

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. 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. Combined with comparative analysis and functional validation, we identified a 183 bp ECR (termed tnni1b-ECR183) that was l...Continue Reading

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Citations

May 1, 2021·International Journal of Molecular Sciences·Costantino ParisiCecilia Lanny Winata

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Methods Mentioned

BETA
transgenic
RNA-seq
PCR
Assay
electrophoretic
Protein
Protein Assay
confocal microscopy
exome sequencing

Software Mentioned

ImageJ
EMSA
JASPAR
Adobe Illustrator
ECR Browser
PROMO

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