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Transient and transgenic analysis of the zebrafish ventricular myosin heavy chain (vmhc) promoter: an inhibitory mechanism of ventricle-specific gene expression.

ABSTRACT: The zebrafish ventricular myosin heavy chain (vmhc) gene exhibits restricted expression in the ventricle. However, the molecular mechanism underlying this chamber-specific expression is unclear. Here, we exploited both transient and transgenic technologies to dissect the zebrafish vmhc promoter. We demonstrated that a combination of two transient assays in this animal model quickly identified chamber-specific cis-elements, isolating a 2.2 kb fragment upstream from the vmhc gene that can drive ventricle-specific expression. Furthermore, deletion analysis identified multiple cis-elements that exhibited cardiac-specific expression. To achieve chamber specificity, a distal element was required to coordinate with and suppress a proximal enhancer element. Finally, we discovered that Nkx2.5-binding sites (NKE) were essential for this repressive function. In summary, our study of the zebrafish vmhc promoter suggests that ventricle-specific expression is achieved through an inhibitory mechanism that suppresses expression in the atrium. Developmental Dynamics 238:1564-1573, 2009. (c) 2009 Wiley-Liss, Inc.

SUBMITTER: Zhang R

PROVIDER: S-EPMC2756512 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Transient and transgenic analysis of the zebrafish ventricular myosin heavy chain (vmhc) promoter: an inhibitory mechanism of ventricle-specific gene expression.

Zhang Ruilin R Xu Xiaolei X

Developmental dynamics : an official publication of the American Association of Anatomists 20090601 6

The zebrafish ventricular myosin heavy chain (vmhc) gene exhibits restricted expression in the ventricle. However, the molecular mechanism underlying this chamber-specific expression is unclear. Here, we exploited both transient and transgenic technologies to dissect the zebrafish vmhc promoter. We demonstrated that a combination of two transient assays in this animal model quickly identified chamber-specific cis-elements, isolating a 2.2 kb fragment upstream from the vmhc gene that can drive ve ...[more]

PMID: 19322764

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Project description:Studying the importance of genetic factors in a desired cell type or tissue necessitates the use of precise genetic tools. With the introduction of bacteriophage Cre recombinase/loxP mediated DNA editing and promoter-specific Cre expression, it is feasible to generate conditional knockout mice in which particular genes are disrupted in a cell type-specific manner in vivo. In cardiac myocytes, this is often achieved through α-myosin heavy chain promoter (αMyHC)-driven Cre expression in conjunction with a loxP-site flanked gene of interest. Recent studies in other cell types demonstrate toxicity of Cre expression through induction of DNA damage. However, it is unclear to what extent the traditionally used αMyHC-Cre line [1] may exhibit cardiotoxicity. Further, the genotype of αMyHC-Cre(+/-) is not often included as a control group in cardiac myocyte-specific knockout studies. Here we present evidence that these αMyHC-Cre(+/-) mice show molecular signs of cardiac toxicity by 3months of age and exhibit decreased cardiac function by 6months of age compared to wild-type littermates. Hearts from αMyHC-Cre(+/-) mice also display evidence of fibrosis, inflammation, and DNA damage. Interestingly, some of the early functional changes observed in αMyHC-Cre(+/-) mice are sexually dimorphic. Given the high level of Cre recombinase expression resulting from expression from the αMyHC promoter, we asked if degenerate loxP-like sites naturally exist in the mouse genome and if so, whether they are affected by Cre in the absence of canonical loxP-sites. Using a novel bioinformatics search tool, we identified 619 loxP-like sites with 4 or less mismatches to the canonical loxP-site. 227 sites overlapped with annotated genes and 55 of these genes were expressed in cardiac muscle. Expression of ~26% of the 27 genes tested was disrupted in αMyHC-Cre(+/-) mice indicating potential targeting by Cre. Taken together, these results highlight both the importance of using αMyHC-Cre mice as controls in conditional knockout studies as well as the need for a less cardiotoxic Cre driver for the field.

Dataset Information

Transient and transgenic analysis of the zebrafish ventricular myosin heavy chain (vmhc) promoter: an inhibitory mechanism of ventricle-specific gene expression.

Publications

Transient and transgenic analysis of the zebrafish ventricular myosin heavy chain (vmhc) promoter: an inhibitory mechanism of ventricle-specific gene expression.

Similar Datasets

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