Tamoxifen induction of Cre recombinase does not cause long-lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies
The systemic delivery of tamoxifen (Tam) to activate inducible CreERT2-loxP transgenic mouse systems is now widely used in neuroscience studies. This critical technological advancement allows temporal control of DNA-cre recombination, avoidance of embryonically lethal phenotypes, and minimization of residual cell labeling encountered in constitutively active drivers. Despite its advantages, the use of Tam has the potential to cause long-lasting, uncharacterized side effects on the transcriptome and epigenome in the CNS, given its mixed estrogen receptor (ER) agonist/antagonist actions. With the welcome focus on including both sexes in biomedical studies and efforts to understand sex differences, Tam administration could also cause sexually divergent responses that would confound studies. To examine these issues, epigenetic and transcriptomic profiles were compared in C57BL/6 J female and male hippocampus, cortex, and retina 1 month after a 5-day Tam treatment typical for cre induction, or vehicle control (sunflower seed oil). Cytosine methylation and hydroxymethylation levels, in both CG and non-CG contexts, were unchanged as determined by oxidative bisulfite sequencing. Long-lasting Tam transcriptomic effects were also not evident/minimal. Furthermore, there is no evidence of sexually divergent responses with Tam administration and Tam did not alter sex differences evident in controls. Combined with recently reported data that Tam alone does not cause long-lasting changes in behavior and neurogenesis, our findings provide confidence that Tam can be used as a cre-recombinase inducer without introducing significant confounds in transcriptomic and epigenomic neuroscience studies, particularly those focused on genomic and transcriptomic aspects of the aging brain.
KeywordsTransgenic Tamoxifen Sex differences Epigenome Methylation Transcriptome Hippocampus Cortex Retina
The authors acknowledge the Laboratory for Molecular Biology and Cytometry Research at OUHSC for the use of the Core Facility which provided Illumina MiSeq Next Generation Sequencing services and the Oklahoma Medical Research Foundation Clinical Genomics Center which provided NextSeq services Computing for this project was performed at the OU Supercomputing Center for Education and Research (OSCER) at the University of Oklahoma (OU).
This work was supported by grants from the National Institutes of Health (NIH) P30AG050911, R56AG059430, R01AG58430, P20GM125528, R01AG0256161, Veterans Affairs I01BX003906, Oklahoma Center for Adult Stem Cell Research (OCASCR) grant through the Oklahoma Tobacco Settlement Endowment Trust, and Presbyterian Health Foundation.
- Ashpole NM, Logan S, Yabluchanskiy A, Mitschelen MC, Yan H, Farley JA, Hodges EL, Ungvari Z, Csiszar A, Chen S, Georgescu C, Hubbard GB, Ikeno Y, Sonntag WE (2017) IGF-1 has sexually dimorphic, pleiotropic, and time-dependent effects on healthspan, pathology, and lifespan. Geroscience 39:129–145CrossRefGoogle Scholar
- Du M et al (2017) Retinal gene expression responses to aging are sexually divergent. Mol Vis 23:707–717Google Scholar
- Hadad N, Masser DR, Logan S, Wronowski B, Mangold CA, Clark N, Otalora L, Unnikrishnan A, Ford MM, Giles CB, Wren JD, Richardson A, Sonntag WE, Stanford DR, Freeman W (2016) Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice. Epigenetics Chromatin 9:30CrossRefGoogle Scholar
- Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 27:4324–4327CrossRefGoogle Scholar
- Khalaj AJ, Yoon J, Nakai J, Winchester Z, Moore SM, Yoo T, Martinez-Torres L, Kumar S, Itoh N, Tiwari-Woodruff SK (2013) Estrogen receptor (ER) beta expression in oligodendrocytes is required for attenuation of clinical disease by an ERbeta ligand. Proc Natl Acad Sci U S A 110:19125–19130CrossRefGoogle Scholar
- Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, Lucero J, Huang Y, Dwork AJ, Schultz MD, Yu M, Tonti-Filippini J, Heyn H, Hu S, Wu JC, Rao A, Esteller M, He C, Haghighi FG, Sejnowski TJ, Behrens MM, Ecker JR (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341:1237905CrossRefGoogle Scholar
- Malonia SK et al (2011) Gene regulation by SMAR1: role in cellular homeostasis and cancer. Biochim Biophys Acta 1815:1–12Google Scholar
- Mazeyrat S, Saut N, Sargent CA, Grimmond S, Longepied G, Ehrmann IE, Ellis PS, Greenfield A, Affara NA, Mitchell MJ (1998) The mouse Y chromosome interval necessary for spermatogonial proliferation is gene dense with syntenic homology to the human AZFa region. Hum Mol Genet 7:1713–1724CrossRefGoogle Scholar
- Morales-Otal A, Retana-Marquez S, Ferreira-Nuno A, Velazquez-Moctezuma J (2005) Testosterone levels and histological features of reproductive glands in adult male rats treated neonatally with tamoxifen. Neuro Endocrinol Lett 26:729–732Google Scholar
- Podlutsky A, Valcarcel-Ares MN, Yancey K, Podlutskaya V, Nagykaldi E, Gautam T, Miller RA, Sonntag WE, Csiszar A, Ungvari Z (2017) The GH/IGF-1 axis in a critical period early in life determines cellular DNA repair capacity by altering transcriptional regulation of DNA repair-related genes: implications for the developmental origins of cancer. Geroscience 39:147–160CrossRefGoogle Scholar
- Su CY, Lin TC, Lin YF, Chen MH, Lee CH, Wang HY, Lee YC, Liu YP, Chen CL, Hsiao M (2015) DDX3 as a strongest prognosis marker and its downregulation promotes metastasis in colorectal cancer. Oncotarget 6:18602–18612Google Scholar
- van Leeuwen FE, van den Belt-Dusebout AW, van Leeuwen FE, Benraadt J, Diepenhorst FW, van Tinteren H, Coebergh JWW, Kiemeney LALM, Gimbrère CHF, Otter R, Schouten LJ, Damhuis RAM, Benraadt J, Bontenbal M (1994) Risk of endometrial cancer after tamoxifen treatment of breast cancer. Lancet 343:448–452CrossRefGoogle Scholar
- Wen L, Li X, Yan L, Tan Y, Li R, Zhao Y, Wang Y, Xie J, Zhang Y, Song C, Yu M, Liu X, Zhu P, Li X, Hou Y, Guo H, Wu X, He C, Li R, Tang F, Qiao J (2014) Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain. Genome Biol 15:R49CrossRefGoogle Scholar