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Variants in an Hdac9 intronic enhancer plasmid impact Twist1 expression in vitro

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Abstract

Skin tumor susceptibility 5 (Skts5) was previously mapped to mouse chromosome 12 through linkage analysis of skin tumor susceptible Mus musculus (NIH/Ola-S) and skin tumor resistant outbred Mus spretus (SPRET/Out-R) mice. Hdac9 was identified as a potential candidate for Skts5 based on conserved non-synonymous sequence variants and expression analyses. Studies by others identified an enhancer in human HDAC9 that correlated with TWIST1 expression. We identified 45 sequence variants between NIH/Ola-S and SPRET/Out-R mice from the orthologous region of the human HDAC9 enhancer. Variants mapping to intron 18 differentially affected luciferase expression in vitro. NIH/Ola-S clones showed an approximate 1.7-fold increased luciferase expression relative to vector alone or the equivalent clones from SPRET/Out-R-R. Furthermore, cells transfected with a portion of the NIH/Ola-S intron induced 2.2-fold increases in Twist1 expression, but the same region from SPRET/Out-R mice resulted in no up-regulation of Twist1. In silico transcription factor analyses identified multiple transcription factors predicted to differentially bind NIH/Ola-S and SPRET/Out-R polymorphic sites. Chromatin immunoprecipitation studies of two transcription factors, Gata3 and Oct1, demonstrated differential binding between NIH/Ola-S and SPRET/Out-R plasmids that corroborated the in silico predictions. Together these studies provide evidence that the murine orthologous region to a human HDAC9 enhancer also acts as a transcriptional enhancer for mouse Twist1. As ectopic sequence variants between NIH/Ola-S and SPRET/Out-R differentially impacted luciferase expression, correlated with Twist1 expression in vitro, and affected Gata3 and Oct1 binding, these variants may explain part of the observed differences in skin tumor susceptibility at Skts5 between NIH/Ola-S and SPRET/Out-R.

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Acknowledgments

We thank the Parvin laboratory for use of their Fisher Scientific Sonic Dismembrator Model 500. The OSU Comprehensive Cancer Center Nucleic Acids Shared Resource provided support for Sanger Sequencing and qPCR. This work was supported in part by the National Institutes of Health (1R01 CA134461-01), the American Cancer Society (RSG-07-083-1-MGO), and the OSU Comprehensive Cancer Center. TES was supported by a Pelotonia Undergraduate Research Fellowship, and MMG was supported by a Pelotonia Graduate Research Fellowship.

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Authors and Affiliations

  1. Biomedical Sciences Program, The Ohio State University College of Medicine, Columbus, OH, 43210, USA

    Tyler E. Siekmann

  2. Biomedical Sciences Graduate Program, The Ohio State University College of Medicine, Columbus, OH, 43210, USA

    Madelyn M. Gerber

  3. Department of Molecular Virology, Immunology and Medical Genetics and the Division of Human Genetics, Department of Internal Medicine, The Ohio State University Comprehensive Cancer Center, The Ohio State University, 998 Biomedical Research Tower, 460 W. 12th Avenue, Columbus, OH, 43210, USA

    Amanda Ewart Toland

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  1. Tyler E. Siekmann
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Correspondence to Amanda Ewart Toland.

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Siekmann, T.E., Gerber, M.M. & Toland, A.E. Variants in an Hdac9 intronic enhancer plasmid impact Twist1 expression in vitro. Mamm Genome 27, 99–110 (2016). https://doi.org/10.1007/s00335-015-9618-3

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