Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity.
The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR 369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR 369larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously. The vertebrate hypothalamus-pituitary-adrenal (HPA) axis orchestrates physiological, behavioral, and metabolic adjustments required for homeostasis, by dynamically regulating production and secretion of adrenal steroids known as glucocorticoids. In humans the primary glucocorticoid is cortisol, the biological activity of which is mediated by two regulatory proteins in the nuclear receptor family, the ubiquitous glucocorticoid receptor (GR) and the more tissue-restricted mineralocorticoid receptor (MR). The GR binds cortisol less avidly than the MR and is thus more dynamically regulated over the normal physiological range of cortisol fluctuations 1,2. The GR and MR function both as transcription factors and as non-nuclear signaling proteins, including in the central nervous system where both proteins are highly expressed 1-5. Given that the GR is more widely expressed and more dynamically regulated by cortisol, it is generally thought to be the principal mediator of cortisolinduced genomic responses to circadian rhythms and acute stress 5. An important question for understanding GR function is what downstream transcriptional regulatory genes does it regulate, and to what end? Answering this question is not only important for understanding the physiological function and regulation of the GR, but also for deciphering the gene regulatory networks that orchestrate adaptive developmental programming in response to chronic glucocorticoid exposure such as occurs with chronic early life stress 6. The zebrafish has recently emerged as a model system well-suited to investigating the developmental functions of glucocorticoid signaling and mechanisms underlying stress-induced developmental programming 7-...
We describe a real-time (rt) PCR-based method of quantifying DNA damage, adapted from the long-run rtPCR method of DNA damage quantification (LORD-Q) developed by Lehle et al. (Nucleic Acids Res 42(6):e41, 2014). We show that semi-long run rtPCR, which generates amplicons half the length of those generated in LORD-Q, provides equivalent sensitivity for detecting low lesion frequencies, and better sensitivity for detecting high frequencies. The smaller amplicon size greatly facilitates PCR optimization and allows greater flexibility in the use of detection dyes, and a modified data analysis method simplifies the calculation of lesion frequency. The method was used to measure DNA damage in the nuclear and mitochondrial genomes of different tissues in zebrafish of different ages. We find that nuclear DNA damage generally increases with age, and that the amount of mitochondrial DNA damage varies substantially between tissues, increasing with age in liver and brain but not in heart or skeletal muscle, the latter having the highest levels of damage irrespective of age.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-017-2593-x) contains supplementary material, which is available to authorized users.
Objective: Chronic early life stress can affect development of the neuroendocrine stress system, leading to its persistent dysregulation and consequently increased disease risk in adulthood. One contributing factor is thought to be epigenetic programming in response to chronic cortisol exposure during early development. We have previously shown that zebrafish embryos treated chronically with cortisol develop into adults with constitutively elevated whole-body cortisol and aberrant immune gene expression. Here we further characterize that phenotype by assessing persistent effects of the treatment on cortisol tissue distribution and dynamics, chromatin accessibility, and activities of glucocorticoid-responsive regulatory genes klf9 and fkbp5. To that end cortisol levels in different tissues of fed and fasted adults were measured using ELISA, open chromatin in adult blood cells was mapped using ATAC-seq, and gene activity in adult blood and brain cells was measured using qRT-PCR. Results: Adults derived from cortisol-treated embryos have elevated whole-body cortisol with aberrantly regulated tissue distribution and dynamics that correlate with differential activity of klf9 and fkbp5 in blood and brain.
An intact mTERT N-terminus which contributes to mTR binding, DNA binding and telomerase activity is necessary for elongation of short telomeres and the maintenance of functional telomeres. It is reasonable to speculate that relative levels of mTERT-Insi1 may regulate telomere function in specific tissues.
Telomeres have emerged as a promising and important factor modulating cellular and organism responses to ionizing radiation (IR). Pin2/TRF1 interacting protein X1 (PinX1) is an intrinsic telomerase inhibitor and a putative tumor suppressor gene in human cancers. The aim of this study is to investigate the role PinX1 in osteosarcoma (OS) radioresistance. A telomerase-positive OS cell line Saos-2 and a telomerase-negative OS cell line U2OS were used. PinX1 shRNA lentiviral vetors were constructed and transfected to cells. PinX1 expression was determined by real-time quantitative PCR (qPCR) and Western blotting. Relative telomere length (RTL) was detected by using qPCR. Flow cytometric analysis was used to detect cell cycle and apoptosis. Radiosensitivity was determined by colony formation assay. Data showed that, PinX1 knockdown resulted in telomere shortening, G1 phase arrest, increased apoptosis and enhanced IR sensitivity both in Saos-2 and U2OS cell lines, regardless of telomerase status. Our study concluded that PinX1 could serve as a novel predictor for radiotherapy response to OS patients, and the pathway of PinX1-mediated telomere stability might represent a new target to improve the radiotherapy effect of OS.
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