(A) Experimental design. Male mice were fed control or low (11%) protein diet from weaning until sexual maturity, then were mated to females that were raised on control diet. Males were removed after one or two days of mating. Livers were harvested from offspring at three weeks, and RNA was prepared, labeled, and hybridized to oligonucleotide microarrays. (B) Overview of microarray data, comparing offspring of sibling males fed different diets – red boxes indicate higher RNA levels in low protein than control offspring, green indicates higher expression in controls. Boxes at the top indicate comparisons between two male (purple) or two female (yellow) offspring. Each column shows results from a comparison of a pair of offspring. Only genes passing the stringent threshold for significant change () are shown. Data are clustered by experiment (columns), and by genes (rows). (C) Validation of microarray data. Quantitative RT-PCR was used to determine levels of Squalene epoxidase (Sqle) relative to the control gene Vitronectin (Vtn) which showed no change in the microarray dataset. Animals are grouped by paternal diet and by sex, and data are expressed as ΔC_T between Sqle and Vtn, normalized relative to the average of control females. Additional validation is shown in . p values were calculated using t-test. See also , .

Comparison of upregulated gene expression profile with a compendium of public datasets of hepatic gene expression. A clustering of our upregulated genes according to their notation in the 28 significant (p<0.00025) overlapping signatures from an assembled compendium of 120 publicly-available murine liver signatures under various conditions and genetic perturbations (GEO, (; ). For each significant profile, the majority of overlapping genes are shown as yellow, while genes with opposite regulation (ie down rather than up in the dataset in question) are blue. The genes divide into two distinct clusters, one enriched in DNA replication and the other in various categories of fat and cholesterol biosynthesis. See also and .

(A) Cholesterol biosynthesis. Genes annotated as cholesterol biosynthesis genes are shown, with colors indicating average difference in expression in low protein vs. control comparisons. (B) Many genes upregulated in the low protein cohort are SREBP targets. Upregulated cluster from is shown, along with data from (). Genes scored as up in both replicates from Ref. () are shown as yellow, genes scored as down are blue. Columns show data from transgenic mice overexpressing SREBP-1a or SREBP-2, or from scap−/− knockout mice. (C) Cholesterol levels are decreased in livers of low protein offspring. Data from lipidomic profiling of liver tissue from three control and three low protein animal are shown as mean +/− standard deviation. Red line indicates no change. p values were calculated using a paired t-test on log-transformed lipid abundance data. Cholesterol esters, CE; phosphatidylethanolamine, PE; free cholesterol, FC; triacylglycerol, TAG; phopshatidylcholine, PC; cardiolipin, CL; phosphatidylserine, PS ; free fatty acid, FA; lysophosphatidylcholine, LYPC; and diacylglycerol, DAG. See also .

Small (<35 nt) RNAs from the livers of eight offspring (four control, four low protein) was isolated and subject to high-throughput sequencing. MicroRNAs that exhibited consistent changes in all four pairs of animals are shown, with average change shown as a bar and individual comparisons shown as points. See also .

(A) Genomic DNA from control and low protein offspring livers was subjected to reduced-representation bisulfite sequencing. For all annotated promoters, average fraction of CpGs that were methylated is shown for the control sample (x axis) compared to the low protein sample (y axis). Red and green dots indicate promoters with significant (p < 0.05) methylation changes of over 10%. (B) As in (A), for nongenic CpG islands. (C) Promoter cytosine methylation changes are uncorrelated with gene expression changes. For each promoter, the average change in cytosine methylation is compared to the change in mRNA abundance from . See also and .

(A) Differential methylation of a putative Ppara enhancer. Top panel shows a schematic of chromosome 15: 85,360,000–85,640,000. Zoomed in region represents chr15: 85,514,715–85,514,920. RRBS data for one control and one low protein offspring pair is shown below, with assayed CpGs represented as boxes colored to indicate % of clones methylated. Numbers to the left indicate % methylation, with number of sequence reads covering the CpG in parentheses. (B) Ppara is downregulated in most low protein offspring livers. Box plot shows mean, quartiles, and highest and lowest values from . (C) Putative enhancer methylation correlates with Ppara downregulation. DNA from eight control and nine low protein pairs of offspring livers was bisulfite treated, and at least 13 clones were analyzed for each animal. % methylation at each of the 12 CpGs in this region plotted on the y axis, data are shown as mean+/−SEM. (D) Individual bisulfite clones are shown for three control and three low protein offspring. White circles indicate unmethylated CpGs, black circles indicate methylated CpGs. Microarray data for change in Ppara RNA levels between the paired animals is shown to the left, in log₂. Values under each bisulfite grouping indicate overall % methylation, with number of clones analyzed in parentheses.

(A) MeDIP-sequencing data is shown for two liver samples (top two tracks), and four sperm samples (bottom four), at a maternally-methylated region (Gnas, left) and a paternally-methylated region (Rasgrf1, right). (B) Comparison of control and low protein methylation. For each promoter, methylation levels were averaged for 8 kb surrounding the TSS, and values are scatterplotted for control sperm (x axis) vs. low protein sperm (y axis). x and y axes are plotted on logarithmic scales (C) As in (B), but for control vs. caloric restriction. (D) As in (B), but for the pair of control samples. Similar results for (B–D) are found when focusing on the 1 kb surrounding the TSS (not shown). See for analyses of consistent RNA and chromatin differences between low protein and control sperm.