Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism

Application of Epigenetic Techniques to Study Lactase Expression

DNA methylation is a highly studied epigenetic modification. Numerous techniques have been developed to investigate DNA methylation and its role in gene regulation. Lactose intolerance has been identified as a simple phenotype, regulated by a single gene. Due to its simple phenotype, lactose intolerance represents an ideal model to investigate the epigenetic mechanisms that govern gene expression. Here, we investigated lactase expression in aging mice, and used molecular inversion probe enrichment followed by DNA sequencing to study epigenetic modifications in segmental duplications and unique regions of the genome associated with the lactose intolerance in humans. Lactase was expressed in a mosaic pattern in the mouse small intestine, and expression significantly decreased in older mice. Molecular inversion probe enrichment of unique genomic regions proved challenging, and did not provide informative data. No associations between lactase expression levels and the DNA methylation profile of the lactase gene in segmental duplications was found.M.Sc

Transgenerational Effects of Antenatal Synthetic Glucocorticoid Exposure on Transcription and Methylation in the Developing Brain

The early environment has long term influences on future health that are heritable over multiple generations via maternal and paternal lineages. Antenatal synthetic glucocorticoids (sGC) are administered to women at risk for pre-term delivery because they reduce the morbidity and mortality associated with newborn respiratory distress syndrome. However, the administration of multiple courses of antenatal sGC results in long-term programing of behavioral and physiological responses to stress. In this research, we performed transcriptomic and epigenomic analyses to ascertain the long-term programming effects of antenatal sGC in the brains of first generation (F1) juvenile males and three generations of juvenile female offspring of the paternal lineage. We hypothesized that antenatal sGC exposure results in transgenerational changes in transcription and DNA methylation in the hypothalamic paraventricular nucleus (PVN), prefrontal cortex (PFC) and hippocampus, due to their role in the regulation of the HPA-axis. We observed that antenatal sGC results in generation-, sex-, and brain region-specific changes in gene expression and DNA methylation in three generations juvenile female offspring of the paternal lineage. In the PVN, exposure to sGC programmed large transgenerational changes in gene expression, including type II diabetes, thermoregulation, and collagen formation gene networks. The PFC was the only brain region to show overlap in the significantly differentially expressed genes across all three generations of juvenile females and F1 males. In the PFC, the expression of four genes that were significantly decreased in sGC offspring explained 20-29% of the variability in locomotor behavioral phenotypes. Epigenetic analyses of the hippocampus identified significant changes in CpG methylation in regulatory regions of small non-coding RNAs involved in transcription and splicing. These findings may implicate alternative splicing as a mechanism involved in the transgenerational transmission of the effects of antenatal sGC. This is the first study to show that antenatal sGC exposure, a clinically relevant treatment, results in transgenerational changes in gene expression, and methylation over three generations, via paternal transmission.Ph.D.2020-07-10 00:00:0

Chorioamnionitis Induces a Specific Signature of Placental ABC Transporters Associated with an Increase of miR-331-5p in the Human Preterm Placenta

Background/Aims: The ATP-binding cassette (ABC) transporters mediate drug biodisposition and immunological responses in the placental barrier. In vitro infective challenges alter expression of specific placental ABC transporters. We hypothesized that chorioamnionitis induces a distinct pattern of ABC transporter expression. Methods: Gene expression of 50 ABC transporters was assessed using TaqMan® Human ABC Transporter Array, in preterm human placentas without (PTD; n=6) or with histological chorioamnionitis (PTDC; n=6). Validation was performed using qPCR, immunohistochemistry and Western blot. MicroRNAs known to regulate P-glycoprotein (P-gp) were examined by qPCR. Results: Up-regulation of ABCB9, ABCC2 and ABCF2 mRNA was detected in chorioamnionitis (p<0.05), whereas placental ABCB1 (P-gp; p=0.051) and ABCG2 (breast cancer resistance protein-BCRP) mRNA levels (p=0.055) approached near significant up-regulation. In most cases, the magnitude of the effect significantly correlated to the severity of inflammation. Upon validation, increased placental ABCB1 and ABCG2 mRNA levels (p<0.05) were observed. At the level of immunohistochemistry, while BCRP was increased (p<0.05), P-gp staining intensity was significantly decreased (p<0.05) in PTDC. miR-331-5p, involved in P-gp suppression, was upregulated in PTDC (p<0.01) and correlated to the grade of chorioamnionitis (p<0.01). Conclusions: Alterations in the expression of ABC transporters will likely lead to modified transport of clinically relevant compounds at the inflamed placenta. A better understanding of the potential role of these transporters in the events surrounding PTD may also enable new strategies to be developed for prevention and treatment of PTD