Donkin et. al. Presented by: Natasha Granneman and Christina Tran Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans Donkin et. al. Presented by: Natasha Granneman and Christina Tran

Background/Importance Obesity has behavioral and heritable causes Children of obese FATHERS have a higher risk of obesity Inherited paternal factors? Socioeconomic status of the father? Nutritional status and exercise have been shown to alter DNA methylation in somatic tissues Can obesogenic factors change gamete epigenomes? Believed to be more than just socioeconomic status because: -studies in rodents & epidemiological data show that paternal nutritional status can directly affect the health of offspring

Background Epigenetics = anything other than the DNA sequence that influences gene expression Epigenetic changes can be heritable!

Hypotheses Spermatozoa from obese men have an altered epigenome relative to lean men Weight loss remodels the epigenome of spermatozoa in human obesity

Methods Lean vs. Obese Collected single ejaculates from 23 Caucasian males 13 lean, 10 obese Gastric Bypass induced weight loss Collected single ejaculates from 6 obese men at 3 time points: 1 week before surgery 1 week after surgery 1 year after surgery

Methods Characterized histone positioning Micrococcal nuclease sequencing (MNase-seq) Compared small non-coding RNA (sncRNA) expression sncRNA-seq Compared DNA methylation patterns Reduced representation bisulfite Seq (RRBS) Genotyped all individuals for SNP’s associated with obesity Infinum CoreExome-24 BeadChip

Results histone positioning was not altered in sperm between lean and obese men lean and obese men had a 2% genome retention rate 5% of enhancer regions reported in somatic cells retained histones in mature sperm

Results Looked at sncRNA content in lean and obese men only expression levels of piRNA changed significantly Predicted targets of the differentially expressed piRNAs were in the categories “Chromatin” and “Chemdependancy” CART = a regulator of food intake involved in obesity CART piRNA= known to be predominantly expressed in the germ line because they maintain genomic stability by repressing transposable elements, and they can inhibit gene expression post-transcriptionally

Results appetite control genes 9,081 genes differentially methylated between lean and obese men mostly nervous system development genes 274 disease related genes were differentially methylated regulators of appetite control genes related to obesity and metabolism obesity/metabolism genes NPY-neuropeptide that increases food intake and fat storage FTO-more copies → higher obesity risk

Results Genotyped 23 known SNPs associated with obesity to see if epigenetic variation between lean and obese could be due to polymorphisms polymorphisms could influence methylation of proximal cytosines Saw that none of the SNPs were within a distance of 1,000 bp of a methylated cytosine Further analysis of SNPs showed no relationship between the SNP and epigenetic variability test whether methylation changed due to something environmental or if it's because a change in the sequence (SNP) altered the affinity of that site for methylation

Results Studied sperm DNA methylation profiles in obese men before and after gastric bypass surgery (GBP) Pre-GBP vs. 1 week after GBP: 1,509 unique genes were differentially methylated suggests that changes can occur in last stages of sperm maturation Pre-GBP vs. 1 year after GBP: 3,910 unique genes were differentially methylated Sperm maturation takes 3 months

Results 2,681 of genes with altered DNA methylation between pre-GBP and GBP-1y were also altered in lean vs obese indicates methylation hotspots that coincide with nutritional intake Many of these genes can have SNPs associated with severe onset obesity, but none of the participants had an obesity SNP overlap was not significant to equate the 2 groups as having the same effect on methylation, but can indicate genes that are differentially methylated in response to nutrition none of the participants had an obesity-carrying SNP, showing gbp induced weight loss modulates the epigenome of sperm in genes that are master regulators of obesity

Results Compared changes in methylation in sperm and somatic tissues (blood and adipose) before and 1 year after GBP Methylation patterns are unique to each tissue in the 60 CpG sites investigated Environmental factors associated with GBP induce changes in methylation that are unique to spermatozoa

Conclusion Epigenomes of spermatozoa in lean and obese men differ in DNA methylation patterns and piRNA expression Methylome of sperm is altered after GBP surgery the degree to which the sperm epigenome is transmitted to the offspring’s metabolic tissues is unknown Could not determine which specific factors associated with obesity (BMI, glucose, insulin, LDL, HDL, triglycerides, leptin, etc.) were contributing to changes in methylation

Limitations Small sample sizes Some findings were not significant 13 lean, 10 obese, 6 GBP Some findings were not significant Genes “differentially methylated” in lean and obese men (NPY, BDNF, CHST8, etc.) did not have statistical significance Only methylation changes were studied in the GBP cohort Overlap between methylation changes in Lean vs. Obese cohort and pre-GBP vs. GBP-1y cohort was not significant, but claimed the overlap indicates “hotspots for methylation changes in the genome associated with changes in nutritional intake”

References/Additional Reading Donkin, I., Versteyhe, S., Ingerslev, Lars R., Qian, K., Mechta, M., Nordkap, L., Mortensen, B., Appel, Emil Vincent R., Jørgensen, N., Kristiansen, Viggo B., et al. (2016). Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans. Cell Metabolism, in press Brøns, C., Jensen, C.B., Storgaard, H., Hiscock, N.J., White, A., Appel, J.S., Jacobsen, S., Nilsson, E., Larsen, C.M., Astrup, A., et al. (2009). Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men. J. Physiol. 587, 2387–2397. Soubry, A., Murphy, S.K., Wang, F., Huang, Z., Vidal, A.C., Fuemmeler, B.F., Kurtzberg, J., Murtha, A., Jirtle, R.L., Schildkraut, J.M., et al. (2013). Newborns of obese parents have altered DNA methylation patterns at im- printed genes. Int. J. Obes. 39, 650–657.