Location: Houston, Texas2011 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 regulated modification of DNA that affects cell-type specific gene expression. In Obj. 1 we studied a 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 is central to regulation of food intake and energy expenditure. We measured hypothalamic DNA methylation in over 20 genes known to play a role in brain development or hypothalamic regulation, and found several differences in SL vs. C mice at weaning. In Obj. 2 we used a genome-wide method (MSAM) to screen for persistent changes in hypothalamic DNA methylation in SL vs. C mice, but no significant differences were detected. Methylation changes induced by SL exposure must either be too small to be detected by MSAM or occur only in specific cell types in the hypothalamus. Two major brain cell types are neurons and glia. We tested if DNA methylation differs between these cell types; we sorted nuclei from hypothalamic neurons and glia, and performed MSAM. We found differences in gene-specific DNA methylation in neurons and glia. Our future analyses of the effect of early nutrition on hypothalamic DNA methylation will consider these two cell types separately. In Obj. 4 we profiled DNA methylation across the genome to compare androgenetic hydatidiform moles (abnormal placental tissue that contains only DNA inherited from the father), ovarian teratomas (an abnormal growth of unfertilized oocytes in the ovary) and normal placentas. We used a DNA methylation chip that tests 27,000 genomic regions and found that we need to study more samples to find new imprinted genes. We will now use a new array that tests 450,000 genomic regions. We studied the function of NLRP7 to confirm in various cell lines that this protein interacts with CTCF and YY1, two proteins important in the regulation of imprinting, as well as with NPM1, a protein that we found to bind to NRLP7. In Obj. 5 we performed gene expression profiling on a genechip using RNA from liver and muscle of mice born to mothers fed a low-protein diet. We tested two types of muscle: soleus at 21 days of age, and soleus and tibialis anterior at 1 year. We did not find differences, but did find many expression changes in liver at age 1 year. Genes with confirmed altered expression did not show changes in DNA methylation at their promoters. In Obj. 6 we completed MSAM to compare methylation in human embryonic stem cells before and after induced differentiation and identified 3% of genomic regions that are targets for programmed methylation. The results provide specific genomic regions to study underlying regulatory mechanisms. We validated gene expression assays for 10 candidate genes. In Obj. 7, we are developing a novel mouse model to understand the signals that determine locus-specific DNA methylation in vivo. We established a method to further screen and confirm our genetic engineering approach. The ADODR monitors project activities by visits, review of purchases of equipment, review of ARS-funded foreign travel, and review of ARS funds provided through the SCA.
1. A possible role for cohesins in the developmental origins of health and disease. Developmental exposure to adverse environments, such as poor maternal nutrition, has long-term effects on gene expression that influence overall adult health and susceptibility to disease. In Houston, TX, Children's Nutrition Research Center researchers have demonstrated that livers of adult mice that had been born to mothers fed a low protein diet show significant changes in expression of genes encoding a class of proteins called cohesins. Cohesins play a role in how DNA is organized in the nucleus and how certain controlling regions of DNA interact with the genes they control. This exciting new finding suggests a previously undiscovered mechanism by which early nutritional exposures can induce persistent changes in gene expression. These findings help scientists unravel the biological impact of poor nutrition early in life can have in adulthood.