Identification And Characterization Of The Cis-Regulatory Elements Of Teleost Tnnc1a Genes

Objective: The development of multi-chamber heart is closely related to the regulation of multi-gene expression.Troponin C,one of the proteins that regulate muscle contraction,regulates muscle contraction by forming the troponin complex with troponin I and troponin T to modulate the so-called cross bridge interactions between myosin and actin filaments.In teleost,there are two tnnc1 genes existed due to a teleost-specific gene duplication during evolution,with tnnc1 a being expressed in cardiac muscles and tnnc1 b in both the cardiac and slow-twitch skeletal muscles.These differential expression patterns contribute directly to the differences in contractile properties among different types of striated muscles.The tissue-specific expression of genes is usually determined by its transcriptional regulatory factors.Despite the progress made in understanding the transcriptional regulation of mammalian tnnc1,the transcriptional regulation of teleost fish tnnc1 a and tnnc1 b remains unknown.To gain insights into the cardiac expression of c Tn C in teleost,we focused on investigating the transcriptional regulation of tnnc1 a in this study.Methods: Medaka tnnc1 a enhancer was obtained by bioinformatics predictive tools and verified by in vivo transgenic method.Zebrafish tnnc1 a expression pattern was examined through in situ hybridization.Tnnc1 a core promoter and other tnnc1 a enhancers were identified through comparative genetics and their activities was examined by in vivo transgenic method.Tnnc1 a minimal enhancer region was identified through site-specific deletion mutation.In this study,the following experiment were carried out.(1)The known enhancer motifs EM2 and EM4 were used to search the enhancer candidates in medaka genome,the obtained enhancer candidates were tested by in vivo transgenic method.(2)The expression pattern of tnnc1 a m RNA in zebrafish larva was detected by in situ hybridization,especially in the heart.(3)The sequences of teleost tnnc1 a gene were compared and analyzed by UCSC Genome Browser,and the highly conserved non coding elements(CNEs)were selected as tnnc1 a enhancer candidates.The above candidates were identified through in vivo transgenic methods.(4)The tested enhancer sequences with transcriptional activity were segmented by site-specific deletion mutation to find the minimal fragment that plays the most core role in transcriptional regulation.Results:(1)Through prediction and in vivo transgenic method,zebrafish tnnc1 a core promoter region was identified in the region from-108 to +218(taking the transcription start site as +1).The enhancer of zebrafish tnnc1 a gene is located in a 1658 bp fragment in the first intron,and the enhancers of medaka tnnc1 a gene are located in the 1050 bp upstream sequence and a 1649 bp sequence in the first intron.The transcriptional activities of these enhancers only exist in cardiomyocytes.(2)In situ hybridization shows that zebrafish tnnc1 a m RNA was specifically expressed in zebrafish heart,including heart atrium and ventricle.(3)The minimal fragment of tnnc1 a enhancers were identified by site-directed deletion mutation,zebrafish tnnc1 a enhancer was reduced to 509 bp,and medaka tnnc1 a enhancers were reduced to 241 bp in the upstream sequence and 668 bp in the first intron.(4)Motifs and transcription factor binding sites that may functioning in regulating tnnc1 a gene expression were predicted by bioinformatics methods.After sequence homology comparison among teleost species,the highly conserved regions of promoter and enhancer were submitted to JASPAR databases,and the transcription activating elements and heart specific transcription factor binding sites were searched.We found that SP-1 and TATA box which were related to transcription activation,were highly conserved in the core promoter region.Besides,several transcription factor binding sites such as MEF2C、Hey2、Nkx-2.5 were highly conserved in the enhancer regions.Conclusion: Through these experiments,we identified the expression pattern of zebrafish tnnc1 a and the corresponding core promoter and enhancer regions.Besides,MEF2C、Hey2、Nkx-2.5 may be the most key regulatory element to regulate tnnc1 a gene transcription.