Transgenerational effects of stress targeting the 45S rRNA for genetic and epigenetic reprogramming Free

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Paternal experiences, as well as exposures, are suspected to contribute to disease risk in offspring, including cancer. Chromium(III), a glucose regulator, physiological stressor and a preconceptional carcinogen in mice, and acid saline vehicle, which is also a stressor after i.p. injection, were used to examine transgenerational effects on the 45S ribosomal RNA gene, previously identified as a target. Allele frequencies of the rRNA spacer and gene promoters, and DNA methylation in the spacer promoter were quantified by pyrosequencing after polymerase chain reaction amplification. In the sperm of treated mice, both chromium(III) and acid saline induced hypomethylation (paired T test, p<0.0001) and altered frequencies in 2 of 5 alleles (unpaired T test, P<0.05) in reference to untreated group. We further monitored these genetic and epigenetic changes over several developmental stages, including day-8 (10 litters each) and day-11 (9 to10 litters each) embryos, and three tissues (lung, liver, and sperm from epididymis) of 6-week adults (7 to 9 litters each) in the offspring. The trend of differential DNA methylation across 7 CpG sites disappeared in day-8 embryos but emerged to distinct patterns according to gender at day-11 embryonic stage, displaying unique patterns for the acid saline group in males and the chromium(III) group in females. This gender-dependent differential methylation widened in 6-week offspring and reached statistically significant level for all 7 CpG sites (unpaired T test, P<0.05), especially for male lung, after correction for any litter effect. The same trend was observed for liver and sperm of 6-week adult. New patterns for frequency of the 5 alleles were also established in both genders and distinguished the chromium(III) group from others. Thus the paternal exposure to stressors led to transgenerational genetic and epigenetic reprogramming of the rRNA gene. This reprogramming appeared to be continuous throughout development and may alter the function of the rRNA, known to be essential for cell growth and proliferation.