A low-protein paternal diet alters hepatic gene expressions in progeny

Back in October I blogged about a recent paper by Ng et al. suggesting evidence for paternal programming of genes passed to offspring.  Overall, the study wasn’t very convincing (in my humble opinion).

But recently Carone et al. give some more evidence that the paternal diet influences the offspring, independent of the maternal diet.

Male mice were raised on a control diet with 20% protein, or a “low-protein” diet of 11% protein and the 9% made up with sucrose.  Importantly, the authors noted that the dietary change that was responsible for the effects on the offspring could be (other than reducing the protein content) from the difference in sucrose, the fat/protein ratio, or others.  These males were then mated with females all raised on the 20% protein control diet, and removed after 1-2 days to reduce their influence on the offspring.  When the offspring were 3 weeks old, they were sacrificed and gene expression differences in the livers of 26 pairs of the control and low-protein groups analyzed by DNA microarrays.

Of 1595 genes with significant differences, 445 were strongly dependent on the paternal diet.  As the study actually took place at 3 different facilities, with 13 litters, over the course of 5 years, the chances for experimental artifacts (because of e.g. circadian cycle, litter size, sacrifice order, cage location, etc) was reduced.

Quantitative real time PCR validated the microarray results.  Squalene expoxidase (Sqle), which is involved in sterol biosynthesis in the low-protein group was increased ~3-fold compared to the controls.

By analyzing Gene Ontology enrichments, it was found that genes involved in fat and cholesterol biosynthesis as well as DNA replication were unregulated, and some genes involved in sequence-specific DNA binding, ligand-dependent nuclear receptor activity were downregulated in the low-protein group.

Taking the low-protein group expression data, they compared it with 120 public rodent liver gene expression sets, of which 28 of these had changes that overlapped with the current study, including profiles from alterations of genes regulate cholesterol and lipid metabolism: SREBP, KLF15, PPARalpha, and ZFP90.  Rodents with mutations that affect growth hormone (GH) & insulin-like growth factor-1 (IGF-1) also matched the low-protein offspring’s liver gene expressions.  These data comparisons confirmed that genes involved in DNA replication and lipid/cholesterol biosynthesis were most linked, and other transcription and growth factors suggest that multiple pathways are affected by a low-protein paternal diet.

The authors found that gene expression changes in the offspring do not mimic changes that occur in the fathers in response to a low-protein diet, rather immune response and apoptosis-related genes were upregulated and those involved in carboxylic acid metabolism were downregulated.

To verify that gene expression changes related to changes in lipid levels, 3 pairs of control and low-protein mice were analyzed for a number of lipid metabolites.  The low-protein group’s livers were depleted of cholesterol & cholesterol esters, but had increases in saturated cardiolipins, saturated free fatty acids, and saturated & monounsaturated triglycerides.

The authors examined microRNAs, as they might be driving the epigenetic differences.  They found of most importance, miR-21, let-7, miR-199, and miR-98 were upregulated in the low-protein diet and miR-210 downregulated.  However, no statistically significant relationships between the miRNAs and gene expression changes were observed (changes were small).  These miRNA may be changed in response to reprogramming and not the instigators.

Next, they checked for another epigenetic change: cytosine methylation.  Bisulfite sequencing showed small changes (10-20%) in CpG methylation between the low-protein and control animals.  Of note, promoter methylation did not globally correlate with gene expression, which the authors explain means that expressions are not epigenetically changed at each gene, rather gene expressions can be widely altered by changes to a small number of upstream regulators.  Of interest, they found a 30% increase in methylation upstream of PPARalpha (likely an enhancer)- suggesting of course a silencing of the enhancer and explaining the reduced expression of PPARalpha observed in the low-protein group.  Interestingly, the gene expression profile of these mice patches that observed in a PPARalpha knockout model, suggesting that the single methylation of PPARalpha could explain a significant amount of the changes in gene expressions observed by the low-protein diet.

Focusing further on this region, they sequenced 17 more animals (8 controls and 9 low-proteins) and found at several CpGs on this locus the methylation differences can vary dramatically.

Next, they checked if the paternal diet alters cytosine methylation in sperm.  Comparing sperm between the controls and low-protein dieters (sorry to the researcher who handled that job), they found that PPARalpha enhancer methylation, nor global cytosine methylation was not different.  The authors suggest that RNA and chromatin would need to be examined to see if they are the carries of epigenetic information.  So they did this next via microarray, and found that both low-protein and a new calorie restricted animal had more “sperm-like” RNA (vs epididymis RNA- I assume they just ground up the whole tissue for analysis) though they were unable to completely rule out contamination issues and RT-PCR could not verify by statistical significance 2 genes of interest- Smarcd3 (chromatin remodeler) & PPARdelta.

The authors further explored the “key epigenetic histone modification H3K27me3 in sperm” and found that it consistently decreased in the low-protein group at the promoter of Maoa (monoamine oxidase) and Eftud1.  This gives a possible epigenetic mechanism for how the low-protein diet is at least partially causing the changes in the offspring.

Summary/Conclusions

A paternal “low-protein” diet altered gene expressions (mainly involved in lipid and cholesterol biosynthesis and DNA replication) in the livers of offspring even though the maternal diet was unaltered.  Through this study, it was found that PPARalpha may be the main mediator of these changes.  As the authors note, PPARalpha is also altered by the maternal diet, so it may be “a key nexus that integrates ancestral dietary information to control offspring metabolism.”

This study also found that DNA methylation was unlikely to be the epigenetic change in the sperm in the fathers that altered the gene expressions in the offspring, rather more likely a change of chromatin packaging (in this study, on H3K27me3 at Maoa).

Because research now shows that both maternal and paternal diets may effect disease risk on subsequent generations, the authors state that we may need to rethink “basic practices in epidemiological studies of complex diseases such as diabetes, heart disease, or alcoholism. We believe that future environmental exposure histories will need to include parental exposure histories as well as those of the patients to disentangle induced epigenetic effects from the currently sought genetic and environmental factors underlying complex diseases.”

Things just get more complex!

This is a really neat example of how bioinformatics can greatly help find complex gene-diet relationships and one of the few convincing studies of a diet-induced paternal influence on offspring.

Reference

Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Meissner A, Weng Z, Hofmann HA, Friedman N, & Rando OJ (2010). Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell, 143 (7), 1084-96 PMID: 21183072