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A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†.

ABSTRACT: Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the -186/-135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (-91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.

SUBMITTER: Urekar C

PROVIDER: S-EPMC6863968 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†.

Urekar Craig C Acharya Kshitish K KK Chhabra Preeti P Reddi Prabhakara P PP

Biology of reproduction 20191001 4

Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 ...[more]

PMID: 31290539

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Project description:BackgroundSeveral 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.MethodsEvolutionary 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.ResultsCombined 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.ConclusionsAn 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.

Dataset Information

A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†.

Publications

A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†.

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