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United States Department of Agriculture

Agricultural Research Service


Location: Children's Nutrition Research Center

2012 Annual Report

1a.Objectives (from AD-416):
Obj 1: Identify genes that show epigenetic dysregulation in obesity using a candidate-gene approach. Subobj 1A. Characterize developmental establishment of DNA methylation at hypothalamic genes known to affect food intake regulation. Subobj 1B. Compare DNA methylation and expression of these genes between lean and obese mice. Obj 2: Determine if methylation and expression of specific genes in hypothalamus and/or adipose tissue differ between lean and obese mice. Subobj 2A. Identify genomic loci in hypothalamus and adipose tissue at which epigenetic dysregulation is associated with obesity. Subobj 2B. Determine if interindividual epigenetic variation at these loci is found before the onset of obesity. Obj 3: Determine if maternal obesity and/or nutrition before and during pregnancy persistently alters epigenetic regulation in offspring hypothalamus or adipose tissue. Subobj 3A. Identify genomic loci at which epigenetic dysregulation is induced by maternal obesity. Subobj 3B. Determine if this induced epigenetic dysregulation can be prevented by altering maternal diet. Obj 4: Identify placental epigenetic mechanisms that affect fetal nutrition, growth and development. Subobj 4A. Use methylation-specific amplification in conjunction with MSAM of genomic DNA extracted from trophoblast of normal term placentas and androgenetic complete hydatidiform moles to identify novel imprinted genes and other epigenetically regulated genes in trophoblast that play a role in regulation of fetal nutrition. Subobj 4B. Analyze how NLRP7, which is mutated in women with biparental hydatidiform moles, contributes to imprinting in early embryo and placenta. NLRP7 protein, known to have a role in innate immunity, may be a link between environmental changes (such as suboptimal maternal nutrition) and imprinting alterations during development. Obj 5: Determine how programming of glucose intolerance, obesity, and epigenetic dysregulation of skeletal muscle-growth in mice is affected by maternal diet during development. Preliminary data show that offspring of mice exposed to maternal low-protein (MLP) diet have reduction in skeletal muscle mass at <1 yr of age and possibly glucose intolerance. Subobj 5A. Extend phenotypic studies on muscle development, glucose tolerance, growth and obesity to include maternal methyl-donor depleted (MDD) diets and to follow offspring exposed to MLP or MDD diets for up to 18 months. Subobj 5B. To find the molecular basis for the altered muscle phenotype after MLP diet, we will perform array-based gene expression and methylation profiling, as well as gene expression analysis and DNA methylation analysis of candidate genes in hindleg muscle-tissues from 21-d-old and 1-yr-old mice exposed to MLP diet. Obj. 6 Utilize genome-wide DNA methylation profiling to determine if epigenetic programming/reprogramming contribute to lineage-specific patterns of gene expression. Obj. 7 Develop a targeted knock-in mouse model.

1b.Approach (from AD-416):
Children's Nutrition Research Center scientists will use the agouti viable yellow (Avy) mouse model to study the effects of maternal obesity on the risk of obesity in the offspring. DNA methylation in two tissues is known to play important roles in body weight regulation, hypothalamus and adipose tissue. Scientists will study this by using both a candidate gene approach and a genome-wide DNA methylation profiling technique (MSAM). In human studies, our research team will use MSAM and commercially available methylation screening tools such as the Illumina Infinium methylation array to identify genomic regions of differential methylation in complete hydatidiform moles compared with normal term placentas. Also, to characterize the role of NLRP7 in genomic imprinting, we will use chromatin immunoprecipitation to assess interactions of NLRP7 with chromatin, identify DNA binding sites of NLRP7 by electromobility shift assays, and screen for NLRP7 protein binding partners using yeast two-hybrid assays. Lastly, previous studies in a mouse model of the effects of maternal low protein diet on skeletal muscle development in the offspring will be extended to include longer-term studies and additional types of early exposures. This research will investigate fundamental mechanisms regulating DNA methylation during development, and characterize their involvement in nutritional programming during critical ontogenic periods.

3.Progress Report:

DNA methylation is a developmentally regulated modification of DNA that helps control cell-type specific gene expression. In subobjectives 2A and 2B we continued our studies in an established mouse model in which early postnatal overnutrition by suckling in small litters (SL) induces permanent increases in body weight and fatness, relative to mice suckled in normal size control litters (C). The hypothalamus plays a central role in regulation of food intake and energy expenditure. In the previous project period, we used a genome-wide method (MSAM) to screen for persistent changes in hypothalamic DNA methylation in SL vs. C mice. No significant differences were detected. We tested 24 candidate genes for persistent differences in DNA methylation in hypothalamus of SL vs. C mice, and found evidence of such changes at several loci. We submitted a manuscript on these findings. Based on suggestions of the reviewers, we performed gene expression profiling in SL vs. C hypothalamus, and identified several interesting persistent changes related, in particular, to formation of neuronal projections. We are also in the process of integrating the genome-wide methylation data (MSAM) with the expression profiling data. In subobjective 3A, utilizing 'cross-fostering' studies in Avy/a mice, we have demonstrated conclusively that the obesity-promoting effect of maternal obesity occurs during fetal development (not during the suckling period). We subsequently collected hypothalamus from weanling and adult offspring who were born to lean (a/a) and obese (Avy/a) mothers (but all fostered to lean mothers for the suckling period), for assessment of genome-wide DNA methylation. To achieve improved cellular resolution, we have been optimizing methods to study DNA methylation separately in the two major cell types comprising the hypothalamus: neurons (which relay signals) and glia (which provide structure/support for neurons). We found the published methods for sorting neuronal and glial nuclei to have poor reproducibility, and expended considerable effort in the last year troubleshooting and optimizing these. Now we have a robust, reliable, and reproducible method, and are isolating neuronal and glial DNA from hypothalamus of genetically identical offspring of lean and obese mothers (over 20/group). These will be compared by MSA-seq (an improved genome-wide methylation assay that is more quantitative and offers better resolution than MSAM). For subobjective 4A we have continued to work on the methylation array analysis of term placentas and androgenetic complete hydatidiform moles (HM). We found several sites with possible changed methylation. These may control new imprinted genes, which are genes of which only either the copy inherited from the mother or the copy inherited from the father is active. We are now doing experiments to confirm that they are expressed like this in placenta. While some other groups have published on similar experiments, we are working on some genes that were not reported before to be imprinted. If these are indeed imprinted in placenta, this may be important for fetal nutrition. In subobjective 4B we have continued to do experiments to find the protein partners of NLRP7, which we are studying as a cause of recurrent biparental HMs. For this, we have now also studied C6ORF221, another protein implicated in in the same pathology. For subobjective 5 we collected the hypothalamus (a part of the brain that regulates appetite and food intake) of mice that were born to mothers who were fed either a control diet or a low protein diet from before their pregnancies and until the pups were weaned (at 21 days of age) and at 1 year of age. We did gene expression profiling using an array (a gene-expression "Chip"). We found some changes in gene expression on the arrays, but we could only confirm 2 genes at 1 year of age and could not confirm the differences at 21 days of age when we tested 20 genes individually. In subobjective 6 we have completed an integrated analyses based on genome-wide profiling of both DNA methylation and gene expression changes in human embryonic stem cells before and after induced differentiation. Our data demonstrate that DNA methylation plays a direct role in regulating gene expression during cellular differentiation and de-differentiation. We are currently performing detailed functional studies to characterize the differentially methylated regions and to study the underlying regulatory mechanisms. For subobjective 7 we are developing a novel epigenetically engineered mouse model. We used standard targeting method to insert a human cis-element upstream of mouse p16 (a known tumor-suppressor gene) promoter. We confirmed correct targeting by Southern blotting, and we are crossing the chimeric mice onto C57 background for germline transmission. In addition, to confirm the specificity of our targeting approach, we are generating control mice carrying a negative control DNA at the same p16 locus. Our future study will determine whether the knock-in cis-element induces DNA methylation at endogenous p16 promoter in vivo, and whether the knock-in mice develop spontaneous tumors.

Last Modified: 4/23/2014
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