ZFIN ID: ZDB-PUB-190329-6
Identification of a 42-bp heart-specific enhancer of the notch1b gene in zebrafish embryos
Wang, F., Yang, Q., Wu, F., Zhang, Y., Sun, S., Wang, X., Gui, Y., Li, Q.
Date: 2019
Source: Developmental dynamics : an official publication of the American Association of Anatomists   248(6): 426-436 (Journal)
Registered Authors: Li, Qiang, Wang, Xu
Keywords: NOTCH1, enhancer, evolutionary conserved regions, heart, zebrafish
MeSH Terms:
  • Animals
  • Binding Sites
  • Embryo, Mammalian
  • Enhancer Elements, Genetic*
  • Green Fluorescent Proteins/metabolism
  • Homeobox Protein Nkx-2.5/genetics
  • Homeobox Protein Nkx-2.5/metabolism*
  • Myocardium/chemistry*
  • Receptor, Notch1/genetics*
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics
PubMed: 30919514 Full text @ Dev. Dyn.
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ABSTRACT
NOTCH1 plays a key role in the differentiation of ventricles, and mutations are strongly associated with both sporadic and familial bicuspid aortic valves. However, few heart-specific enhancers have been identified to date.
In this study, we investigated evolutionary conserved regions (ECRs) that might act as potential enhancers within the region approximately 150-kb upstream and downstream of the NOTCH1 gene. Functional validation revealed that one 127-bp ECR located ~85-kb downstream of the NOTCH1 gene drives GFP expression in the zebrafish embryo heart. Transcription factor (TF) prediction and core TF distribution analyses were performed to identify the core region. Dissection of ECR3 was performed to identify the 42-bp sequence, which is sufficient for heart-specific expression. In situ hybridization experiments showed that notch1b is expressed in the heart. Overexpression experiments in cells indicated that NKX2-5 is critical for enhancer activity. Mutation of the NKX-5 binding site significantly decreased reporter gene expression. Next, compared with the commonly used myocardium-labeled zebrafish transgenic strain Tg(cmlc2: mCherry), this 42-bp enhancer-labeled stable line mediated a similar expression pattern but with a smaller core region.
This study identified a 42-bp heart-specific enhancer near the NOTCH1 gene and further verified its functional targeting by NKX2-5. This article is protected by copyright. All rights reserved.
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