Parent-of-origin effects propagate through networks to shape metabolic traits
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Evaluation Summary:
This paper will be of interest to scientists studying metabolism and those interested in the evolution of genomic imprinting. The authors show how parent-of-origin effects in crosses between inbred strains of mice can arise from epistasis between imprinted and unimprinted loci. They consider scenarios for the interactions of imprinted and unimprinted genes in adipocytes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Parent-of-origin effects are unexpectedly common in complex traits, including metabolic and neurological traits. Parent-of-origin effects can be modified by the environment, but the architecture of these gene-by-environmental effects on phenotypes remains to be unraveled. Previously, quantitative trait loci (QTL) showing context-specific parent-of-origin effects on metabolic traits were mapped in the F 16 generation of an advanced intercross between LG/J and SM/J inbred mice. However, these QTL were not enriched for known imprinted genes, suggesting another mechanism is needed to explain these parent-of-origin effects phenomena. We propose that non-imprinted genes can generate complex parent-of-origin effects on metabolic traits through interactions with imprinted genes. Here, we employ data from mouse populations at different levels of intercrossing (F 0 , F 1 , F 2 , F 16 ) of the LG/J and SM/J inbred mouse lines to test this hypothesis. Using multiple populations and incorporating genetic, genomic, and physiological data, we leverage orthogonal evidence to identify networks of genes through which parent-of-origin effects propagate. We identify a network comprised of three imprinted and six non-imprinted genes that show parent-of-origin effects. This epistatic network forms a nutritional responsive pathway and the genes comprising it jointly serve cellular functions associated with growth. We focus on two genes, Nnat and F2r , whose interaction associates with serum glucose levels across generations in high-fat-fed females. Single-cell RNAseq reveals that Nnat expression increases and F2r expression decreases in pre-adipocytes along an adipogenic trajectory, a result that is consistent with our observations in bulk white adipose tissue.
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Author Response:
Reviewer #1:
A number of metabolic traits show differences between reciprocal crosses of inbred mouse strains. These can be conceptualized as parent-of-origin effects. Differential expression (DE) at an unimprinted locus can be a pleiotropic side-effect of allele-specific expression (ASE) at an imprinted locus. There is no parent-of-origin effect at the unimprinted locus, in the sense that there would be no change in expression at this locus if the parental origin of the two alleles were reversed while keeping parental origin of alleles at the imprinted locus unchanged. Sexual recombination will have the effect of randomizing alleles at the unimprinted locus relative to alleles at the imprinted locus.
Expression of the imprinted gene Nnat and unimprinted gene F2r were correlated in the author's analyses of …
Author Response:
Reviewer #1:
A number of metabolic traits show differences between reciprocal crosses of inbred mouse strains. These can be conceptualized as parent-of-origin effects. Differential expression (DE) at an unimprinted locus can be a pleiotropic side-effect of allele-specific expression (ASE) at an imprinted locus. There is no parent-of-origin effect at the unimprinted locus, in the sense that there would be no change in expression at this locus if the parental origin of the two alleles were reversed while keeping parental origin of alleles at the imprinted locus unchanged. Sexual recombination will have the effect of randomizing alleles at the unimprinted locus relative to alleles at the imprinted locus.
Expression of the imprinted gene Nnat and unimprinted gene F2r were correlated in the author's analyses of reproductive fat pads from their mice and their interaction was predictive of basal glucose levels. In a single-celled analysis, using an existing database, expression of the paternally-expressed imprinted gene Nnat increases and expression of the unimprinted gene F2r decreases along an adipogenic trajectory in preadipocytes.
One reason why small mammals, such as mice, socialize is to keep warm. Within a huddle or nest, heat generation consumes individual substrates for a communal benefit and this can create selection for imprinted expression when members of groups are asymmetric kin. This conflict has been discussed in the context of the effects of imprinted genes on brown adipocytes that use UCP1 to 'uncouple' oxidative phosphorylation in mitochondria (see Current Biology 18: R172). A UCP1-independent mechanism of non-shivering thermogenesis in skeletal muscle, and beige adipocytes (see Frontiers in Endocrinology 11: 498, involves 'uncoupling' of the SERCA channel that is regulated by Nnat. The role of Nnat is SERCA-dependent thermogenesis is yet to be established.
This is an interesting hypothesis, and one that extends our hypothesis by suggesting that Nnat alters beige thermogenesis, which would alter basal glucose levels. The primary effect would be adipogenic and the secondary effect could be thermogenic, both resulting in altered glucose levels. This would be very interesting to follow-up on in subcutaneous adipose which has been shown to beige in mice. We have added to the text in the discussion.
Reviewer #2:
The authors conducted experiments to examine whether non-imprinted genes interacted with imprinted genes, explored gene pairs that may affect phenotype, and identified two genes, Nnat and Cdkn1c that may initiate parent-of-origin effects. A major strength is the testing of these predictions in a new cohort by manipulating phenotype by diet.
I focus my review on the design. My major concern is the nature of the group assignment to diet, which would require different statistical analysis.
"At three weeks of age, animals were weaned into same-sex cages and randomly placed on high fat (42% kcal from fat; Teklad TD88137) or low-fat (15% kcal from fat; Research Diets D12284) isocaloric diets"
- To be sure I and readers can understand, were the animals first placed into same-sex cages, then the cage was randomly assigned to a diet? If so, the unit of analysis is the cage, and results need to be analyzed as though the animals are not independent within cages. This does not currently seem to be the case, and the analyses not valid.
We thank the reviewer for the comment and we have added detail to the methods for clarity. The animals in each cage for each generation represent animals from different litters. The experimental unit of analysis is the individual animal: for the RNAseq studies one animal from each cage was randomly selected for sequencing in the F1 population. For the F2 animals, animals that were randomly placed into a cage and diet were not genetically identical. Specific details are described in the methods.
- Please describe how many animals were housed per cage and how many cages there were for each diet.
These details have been added to the methods.
- Please describe the method of randomization.
We used a random number generator in R, and have included this detail in the text.
To fully assess the methods and facilitate reproducibility, additional information is needed to describe items as recommended by the ARRIVE guidelines (https://arriveguidelines.org/sites/arrive/files/documents/ARRIVE%20guidelines%202.0%20-%20English.pdf). This includes: the number of animals represented in each experiment/figure (this is not clear throughout the text); the sex of animals used in each experiment; whether order of measurements or animal/cage positioning were randomized (or if not); whether any blinding was performed; housing conditions if available (e.g., room temperature and humidity, light/dark cycle), additional details about diet (whether ad lib, water quality), whether any animals were excluded from analysis after being assigned to a diet; cage type and bedding.
We have added these details to conform to the ARRIVE guidelines as suggested.
Reviewer #3:
Macias-Velasco et al. aimed to demonstrate that non-imprinted genes could generate parent-of-origin effects on metabolic traits, such as glucose concentrations, through interactions with imprinted genes. They used four populations at different levels of intercrossing of inbred mice, and by doing so were able to demonstrate that non-imprinted genes interact with imprinted genes and by doing so impact the animal's phenotype. They focused on two genes in particular, Nnat, and F2r, in high fat-fed female mice as the covariation of these two genes associated with basal glucose concentrations and the relationship. They provided a biological validation of this relationship by demonstrating it consistently across multiple generations. Interactions between imprinted and non-imprinted genes have been previously demonstrated, but the present study took it one step further to identify one possibly reason as to why there is such a prevalence of parent-of-origin effects by detailing the interactions of ASE and DE genes and how that interaction leads to a specific phenotype. The genes the authors identified appear to play a role in adipogenesis. The results may allow for better prediction of an individual's phenotype based on their genome.
The authors conducted a lot of work, and provided a lot of data for the reader, but the paper can be strengthened by the following:
In the results the authors refer to females on a high-fat diet. It would be useful for the reader to put this in a bigger context and explain the implication of diet. It was unclear whether the figures represented sexes combined, and if so, it would have been useful to show the results in males, even if they were null. The authors demonstrated that Nnat expression covaries with F2r in high fat-fed females, yet used available scRNAseq data collected from C57BL/6J epididymal adipose tissue to examine which cell types express these two genes and whether the negative correlation persisted across an adipogenic trajectory (which it did). In mice, the visceral fat pads in the perigonadal region are known as epididymal in males and periovarian in females. Although it was not implicitly stated that these cells were from males, it is presumed by the name of the fat pad. The authors should address possible limitations and considerations related to sex differences, and possibly even strain differences in these comparisons.
The clarity of the methods and materials needs to be improved. Specifically, to allow for others to reproduce the data and to provide greater transparency, in addition to following the eLife guidelines. The authors should follow the ARRIVE guidelines and cite this in the manuscript. With the multiple populations of mice used, it would make it easier on the reader if the sample size and sex were more clearly outlined. Although the authors state that the mice were randomly placed on high-fat or low-fat diet, how the animals were randomized was not outlined. Another possible consideration would be to include a figure outlining the study design, including sample size/sex for the various populations and components of the study. Also, please identify why the sample size was decided, and whether there were any inclusion/exclusion criteria.
We thank the reviewer for the comments and have modified the text for clarity.
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Evaluation Summary:
This paper will be of interest to scientists studying metabolism and those interested in the evolution of genomic imprinting. The authors show how parent-of-origin effects in crosses between inbred strains of mice can arise from epistasis between imprinted and unimprinted loci. They consider scenarios for the interactions of imprinted and unimprinted genes in adipocytes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
A number of metabolic traits show differences between reciprocal crosses of inbred mouse strains. These can be conceptualized as parent-of-origin effects. Differential expression (DE) at an unimprinted locus can be a pleiotropic side-effect of allele-specific expression (ASE) at an imprinted locus. There is no parent-of-origin effect at the unimprinted locus, in the sense that there would be no change in expression at this locus if the parental origin of the two alleles were reversed while keeping parental origin of alleles at the imprinted locus unchanged. Sexual recombination will have the effect of randomizing alleles at the unimprinted locus relative to alleles at the imprinted locus.
Expression of the imprinted gene Nnat and unimprinted gene F2r were correlated in the author's analyses of reproductive …
Reviewer #1 (Public Review):
A number of metabolic traits show differences between reciprocal crosses of inbred mouse strains. These can be conceptualized as parent-of-origin effects. Differential expression (DE) at an unimprinted locus can be a pleiotropic side-effect of allele-specific expression (ASE) at an imprinted locus. There is no parent-of-origin effect at the unimprinted locus, in the sense that there would be no change in expression at this locus if the parental origin of the two alleles were reversed while keeping parental origin of alleles at the imprinted locus unchanged. Sexual recombination will have the effect of randomizing alleles at the unimprinted locus relative to alleles at the imprinted locus.
Expression of the imprinted gene Nnat and unimprinted gene F2r were correlated in the author's analyses of reproductive fat pads from their mice and their interaction was predictive of basal glucose levels. In a single-celled analysis, using an existing database, expression of the paternally-expressed imprinted gene Nnat increases and expression of the unimprinted gene F2r decreases along an adipogenic trajectory in preadipocytes.
One reason why small mammals, such as mice, socialize is to keep warm. Within a huddle or nest, heat generation consumes individual substrates for a communal benefit and this can create selection for imprinted expression when members of groups are asymmetric kin. This conflict has been discussed in the context of the effects of imprinted genes on brown adipocytes that use UCP1 to 'uncouple' oxidative phosphorylation in mitochondria (see Current Biology 18: R172). A UCP1-independent mechanism of non-shivering thermogenesis in skeletal muscle, and beige adipocytes (see Frontiers in Endocrinology 11: 498, involves 'uncoupling' of the SERCA channel that is regulated by Nnat. The role of Nnat is SERCA-dependent thermogenesis is yet to be established.
-
Reviewer #2 (Public Review):
The authors conducted experiments to examine whether non-imprinted genes interacted with imprinted genes, explored gene pairs that may affect phenotype, and identified two genes, Nnat and Cdkn1c that may initiate parent-of-origin effects. A major strength is the testing of these predictions in a new cohort by manipulating phenotype by diet.
I focus my review on the design. My major concern is the nature of the group assignment to diet, which would require different statistical analysis.
"At three weeks of age, animals were weaned into same-sex cages and randomly placed on high fat (42% kcal from fat; Teklad TD88137) or low-fat (15% kcal from fat; Research Diets D12284) isocaloric diets"
- To be sure I and readers can understand, were the animals first placed into same-sex cages, then the cage was randomly …Reviewer #2 (Public Review):
The authors conducted experiments to examine whether non-imprinted genes interacted with imprinted genes, explored gene pairs that may affect phenotype, and identified two genes, Nnat and Cdkn1c that may initiate parent-of-origin effects. A major strength is the testing of these predictions in a new cohort by manipulating phenotype by diet.
I focus my review on the design. My major concern is the nature of the group assignment to diet, which would require different statistical analysis.
"At three weeks of age, animals were weaned into same-sex cages and randomly placed on high fat (42% kcal from fat; Teklad TD88137) or low-fat (15% kcal from fat; Research Diets D12284) isocaloric diets"
- To be sure I and readers can understand, were the animals first placed into same-sex cages, then the cage was randomly assigned to a diet? If so, the unit of analysis is the cage, and results need to be analyzed as though the animals are not independent within cages. This does not currently seem to be the case, and the analyses not valid.
- Please describe how many animals were housed per cage and how many cages there were for each diet.
- Please describe the method of randomization.To fully assess the methods and facilitate reproducibility, additional information is needed to describe items as recommended by the ARRIVE guidelines (https://arriveguidelines.org/sites/arrive/files/documents/ARRIVE%20guidelines%202.0%20-%20English.pdf). This includes: the number of animals represented in each experiment/figure (this is not clear throughout the text); the sex of animals used in each experiment; whether order of measurements or animal/cage positioning were randomized (or if not); whether any blinding was performed; housing conditions if available (e.g., room temperature and humidity, light/dark cycle), additional details about diet (whether ad lib, water quality), whether any animals were excluded from analysis after being assigned to a diet; cage type and bedding.
-
Reviewer #3 (Public Review):
Macias-Velasco et al. aimed to demonstrate that non-imprinted genes could generate parent-of-origin effects on metabolic traits, such as glucose concentrations, through interactions with imprinted genes. They used four populations at different levels of intercrossing of inbred mice, and by doing so were able to demonstrate that non-imprinted genes interact with imprinted genes and by doing so impact the animal's phenotype. They focused on two genes in particular, Nnat, and F2r, in high fat-fed female mice as the covariation of these two genes associated with basal glucose concentrations and the relationship. They provided a biological validation of this relationship by demonstrating it consistently across multiple generations. Interactions between imprinted and non-imprinted genes have been previously …
Reviewer #3 (Public Review):
Macias-Velasco et al. aimed to demonstrate that non-imprinted genes could generate parent-of-origin effects on metabolic traits, such as glucose concentrations, through interactions with imprinted genes. They used four populations at different levels of intercrossing of inbred mice, and by doing so were able to demonstrate that non-imprinted genes interact with imprinted genes and by doing so impact the animal's phenotype. They focused on two genes in particular, Nnat, and F2r, in high fat-fed female mice as the covariation of these two genes associated with basal glucose concentrations and the relationship. They provided a biological validation of this relationship by demonstrating it consistently across multiple generations. Interactions between imprinted and non-imprinted genes have been previously demonstrated, but the present study took it one step further to identify one possibly reason as to why there is such a prevalence of parent-of-origin effects by detailing the interactions of ASE and DE genes and how that interaction leads to a specific phenotype. The genes the authors identified appear to play a role in adipogenesis. The results may allow for better prediction of an individual's phenotype based on their genome.
The authors conducted a lot of work, and provided a lot of data for the reader, but the paper can be strengthened by the following:
In the results the authors refer to females on a high-fat diet. It would be useful for the reader to put this in a bigger context and explain the implication of diet. It was unclear whether the figures represented sexes combined, and if so, it would have been useful to show the results in males, even if they were null. The authors demonstrated that Nnat expression covaries with F2r in high fat-fed females, yet used available scRNAseq data collected from C57BL/6J epididymal adipose tissue to examine which cell types express these two genes and whether the negative correlation persisted across an adipogenic trajectory (which it did). In mice, the visceral fat pads in the perigonadal region are known as epididymal in males and periovarian in females. Although it was not implicitly stated that these cells were from males, it is presumed by the name of the fat pad. The authors should address possible limitations and considerations related to sex differences, and possibly even strain differences in these comparisons.
The clarity of the methods and materials needs to be improved. Specifically, to allow for others to reproduce the data and to provide greater transparency, in addition to following the eLife guidelines. The authors should follow the ARRIVE guidelines and cite this in the manuscript. With the multiple populations of mice used, it would make it easier on the reader if the sample size and sex were more clearly outlined. Although the authors state that the mice were randomly placed on high-fat or low-fat diet, how the animals were randomized was not outlined. Another possible consideration would be to include a figure outlining the study design, including sample size/sex for the various populations and components of the study. Also, please identify why the sample size was decided, and whether there were any inclusion/exclusion criteria.
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