Epigenetic reprogramming of gene promoters in the liver of adult female mice masculinized by testosterone. Mus musculus

Testosterone (T) is known to masculinize the female phenotype of the liver evidenced as upregulated gene expression of male- and downregulated expression of female-predominant genes. To explore the a possibly T epigenetic control, we here screen genome-wide the liver for T-induced changes of the DNA methylation status of gene promoters. by methylated DNA immunoprecipitation and Nimblegen microarrays. Female C57BL/6 mice are treated with T for 3 weeks and, then, T treatment is discontinued for 12 weeks. The T-induced changes found suggest a functional impact since T induces under the same experimental conditions as used here persistent susceptibility to blood-stage malaria of Plasmodium chabaudi. We hypothesize that T epigenetically dys-regulates initial steps of liver-inherent innate immune mechanisms leading to dys-balanced host responses eventually causing the lethal outcome of otherwise self-healing infections. Overall design: Preparation of DNA: DNA was extracted from individual livers by the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), Between 10 to 15 mg of tissue were placed in liquid nitrogen and thoroughly grounded with a mortar. Tissue powder was incubated at 56C degrees for 4 hours in lysis buffer including 12 mAU proteinase K, before adding 0.4 mg RNase A at room temperature for 2 minutes. DNA was precipitated with ethanol and separated by using QIAamp Mini spin columns to finally obtain 8 to 20 microg of high quality DNA per sample. Purity control and quantification were performed with NANODROP 2000 UV-Vis spectrometer (ThermoSCIENTIFIC, Wilmington, USA). Methylated DNA ImmunoPrecipitation (MeDIP): Genomic DNA (1 microg) from each tissue sample was sonicated in a final volume of 200 microl to produce random fragments ranging in size from 300-1,000 bp by the Vibra Cell 75022 Ultrasonic Processor (Novodirect, Kehl, Germany) with a 2-mm tipp. Immunoprecipitation (IP) of methylated DNA was performed with the MeDIP kit (Diagenode, Liege, Belgium) in accordance with the manufacturer´s instructions. In brief, DNA samples at a final concentration of 0.1 microg/micrl were denatured at 95C for 3 min, quickly chilled on ice and finally immunoprecipitated by adding 0.3 microl of manufacturer bead-immobilized anti-5-methylcytosine antibody. Control input samples contained only 20% of that DNA sample used for IP. The bound DNA was separated from the beads by incubation at 65C for 10 min and by using 1 volume of phenol/chloroform/isoamylalcohol (25/24/1). Hybridization of NimbleGen microarrays: Genomic input DNA and the IP DNA were first amplified by Genome Plex Complete WGA Kit (Sigma Aldrich, Hamburg, Germany) as described in the manufacturer´s protocol. Briefly, 30 ng DNA of a 3 ng/microl DNA solution were amplified by 20 PCR cycles using the Amplification Master Mix. Input DNA and IP DNA were labeled in parallel, and 1 microg of each amplified DNA sample were hybridized to NimbleGen (Roche, Basel, Switzerland) Mouse 385 K RefSeq Promoter Arrays containing all known RefSeq genes (NM prefix). The hybridization procedure was applied as recommended by the manufacturer. Array scanning and data analyses: The hybridized arrays were scanned on an Axon 4000B microarray scanner (Molecular Devices, Sunnyvale, CA), and the images were analyzed with Axon GenePix software version 4.1. Image and data analyses were processed with NimbleScan version 2.5 and SIgnalMap version 1.9 software. The methylation ratios between the IP DNA samples and the control input samples were normalized across samples using the quantile method after performing a variance stabilization using log2 scaling. The mean of each promoter feature was determined from three different biological replicates. All data processing and all graphics were performed with in-house developed functions in Matlab. 6 samples were analyzed Cd0, 3 replicates Td0, 3 replicates