Part 1 focused much more on the types of folate, how they get into cells, and the genetic variants that effect folate-related enzymes and their activity. All fine and dandy background information - Folate's most interesting role, to me, is in its variable effects on genomic methylation aka Epigenetics.
Quick background on genomic methylation - different CpG dinucleotides/islands/shores located throughout the genome have the potential to be methylated. This, alongside histone modifications, are responsible for cell type-specific expression patterns, X inactivation in females, silencing of transposable elements and some of the inter-individual variations in expression levels of different proteins that are not explained by coding sequences/other environmental stresses. The thinking is that the methyl groups inhibit transcription factor binding and subsequent expression rates. NOTE: tissues are extremely heterogeneous organs, filled with multiple cell types - methylation levels are expressed as a percentage of the cells within the tissue which exhibit methylation at that site- it's a caveat for anyone reading epigenetics papers to remember that until single cell RNA seq is able to be performed widely, a lot of genomic methylation studies are just looking at correlations between methylation across all cell types of that organ and overall expression - you can gain enough power from sample size and repeated measures to make that correlation seem more like causation.
Within nutritional epigenetics, folate (as well as choline and betaine) can donate methyl groups to remethylate SAM, which is responsible for methylating the DNA Methyltransferases (DNMTs). It stands to reason that at specific CpG's across the genome, a deficiency or overabundance of methyl donors may lead to differential methylation patterns between individuals - these effects can either be laid down during developmentally plastic times (gestation, early infancy/childhood) or potentially, through the simple loss of methylation over time through lack of cell maintenance - I'd like to pause for a moment here to express a thought that I had while doing some Epigenetics research at Johns Hopkins this summer - is the rat a good model for understanding nutritional epigenetics in humans? We had enough power and repeated effect to say that the availability of methyl donors most likely wasn't effecting our outcomes but I always wondered - if a dam's diet is short on methyl donors, is that deficit equally distributed across all 6-14 pups in the litter? is the deficiency only shown in a few pups? is a deficiency drastic enough to show differentially methylated regions (DMRs) even viable in rats? When looking at the pool of maternal methyl donor's direct effect on DMRs, should a single offspring species be used to draw inference for humans? Just a tangential thought I had - curious on others' opinions.
Much of nutrition's impact on epigenetics has focused on gestation and early development. The Developmental Origins of Disease Hypothesis, originally the Barker Hypothesis, argues that environment disturbances, such as low/high caloric intake through pregnancy and excess glucocorticoid exposure, can ultimately effect later disease susceptibility and pathophysiology throughout a lifetime (1) - I highly recommend reading the citation. This hypothesis largely rests upon data that shows individuals born at Low Birth Weight (LBW) are particularly susceptible to the myriad of conditions that fall under the umbrella of Metabolic Syndrome. This has interesting evolutionary implications - the placental/fetal environment may prime the infant for entering an environment of similar quality. If you are born low birth weight, your physiology (pardon my anthropomorphisizing) expects to enter a low caloric environment - however, when a LBW individual enters a world of McDonalds and soda, there is a mismatch between their expected and actual environment. A mechanism for this has been proposed via epigenetic alteration - there is evidence that excess glucocorticoids can set off a signaling cascade that alters these epigenetic modifications (2) and below, I'm going to run through a couple examples that show nutrition's impact.
The Agouti Mice - The Agouti locus is responsible for variable coat color in mice - it is expressed at specific points in development in hair follicles - a retroviral insertion into an exon of the agouti locus allows for transcription via a promotor in the long term repeat of the inserted element (intracisternal A particle) - this mutated allele allows for yellow hair pigmentation, conveying the A^VY genotype (viable yellow-phaeomelanin), compared to the wild type A^w(black-eumelanin) - this allows for an easily stratified system of classification based off of hair color. This is a simplified explanation, and there are a diverse number of strains that exhibit different coat colors. So why does a nutritional scientist care about this locus? As I mentioned before, transposable elements (like the long term repeat inserted into this locus) are methylated - these are generally viewed as being viral and need to be silenced by methylation, if they haven't already accumulated a silencing mutation. Their promoters, however, can act as alternative promoters for nearby genes (this happens with Apolipoprotein C-1). It's essential for 'normal' functioning to keep these regions methylated; this agouti region, however, has come to be included in a class of loci referred to as Metastable epialleles - basically, this means that these areas are subject to variable methylation, susceptible to environmental disturbances, and the changes in methylation state that occur are upheld mitotically - potentially, transgenerationally (3,4). Wolf (5) showed that supplementation of pregnant A^w/A^w mice's diet with methyl donors (choline, betaine, folate) and b12 led to higher % methylation of the LTR promoter, and more of an agouti color type (referred to as pseudo agouti, because the color coat is brown and not black). Dams with a supplemented diet showed an increase in mean methylation at the specific CpG sites located downstream of the insertion. Nutrition appears to be effecting genomic methylation at some loci. This example also helps to answer the side-note question that I had above: the agouti litters showed a lot of subtle variation in coat color - the availability of methyl donors seems to unequivocally effect the different pups.
The above example has a lot of limitations for human studies - mice have multiple pups, the differential methylation is only occurring at a genotype-dependent site with a retroviral insertion - to name a few. And that's why we look to Gambia...
The Gambia Study (6) used Methylation Specific Amplification Microarrays (MSAMs) to identify differentially methylated regions of the genome in peripheral blood leukocytes (PBL) and hair follicles (HF) from 8 healthy Caucasian males. Their methodology sought to identify metastable epialleles - the majority of CpG sites were found to be the same as expected, and using two tissues allowed for researchers to identify DMRs that were due to tissue-specificity. This left 107 genomic loci exhibiting inter-individual differential methylation. This number was then cut down to 40 sites after removing SNP variants and sites located in subtelomeric regions (due to copy number variation/high genetic variability - genotype has been shown to be responsible for differential methylation in past studies). They analyzed 13 of these sites from PBL and HF samples and confirmed interindividual variation within 8 sites. To confirm that these sites are variable across genetically variable populations and maintained throughout the lifetime, post-mortem tissues (liver,kidney,brain) were obtained from biking accidents in a Vietnamese population. 5 of these 8 showed inter-individual variation. The researchers went through great bioinformatic lengths to control as much for genetic variation, but as they note, it's impossible to rule out the potential that differential methylation at these loci is independent of genotype - however, by looking at three of the 5 sites in 23 monozygotic twins (drawback: they only looked at one tissue type - not able to say that the timing of when the methylation differences occurred), they were able to show that the candidate metastable epialleles weren't affected by genotype. This work allowed them to test how environmental influences, in this case maternal nutrition, altered methylation states.
Gambia is a pretty ideal population to study nutritional epigenetics. There's a strong seasonality, with a wet and a dry season, offering different food choices for pregnant mothers to consume. Rainy season infants show lower birthweights and altered susceptibility to disease. Since methyl donors affected methylation profiles in mice, the researchers predicted that seasonality of birth would affect methylation levels. They had assumed that the nutritionally-stressed rainy season would cause reduced methylation at these epialleles but instead found the opposite. Rainy season births showed over 10% increases in methylation at two of the MEs. After finding no effect on control genes, they were able to conclude that nutrition did have a unique effect on these MEs. They even went on to look at whether genetic variability would affect these results - after testing 5 of these ME's in Asian, Caucasian and Gambian individuals, quite genetically distinct populations, they found the same results. In relation to methyl donors, mothers in the rainy season had higher levels of folate - potentially explaining the higher % of methylation.
This Gambian study raises a lot of promise for nutrition's ability to effect these Metastable Epialleles. More work needs to be understood as to when the timing of differential methylation occurs, what other loci are metastable, what makes a specific site a ME, how is this extra folate interacting with SAM and DNMT's to increase methylation at just these MEs, what role are choline/betaine/serine playing in all of this? What role does dietary methyl donor availability play in maintaining Methylation at these sites?
I hope these two examples excite readers that nutrition is a key player in epigenetic plasticity and development. There are numerous other examples, particularly in colon cancer development, where folate plays a key role. The Dutch Famine is another great example of how nutrition may be effecting epigenetic states and altering disease susceptibility. The final (brief) example, as I alluded to on the first post, comes with some controversy regarding maternal folate supplementation policy:
Imprinted genes will probably get its own post at some point, but there is some evidence to show that folic acid intake may lead to aberrant methylation at imprinted loci. Imprinted genes are merely loci that exhibit mono-allelic expression - either the mother or the father's allele is methylated/unmethylated (in imprinting, methylation doesn't necessarily confer reduced expression because promoters aren't what's methylated, imprint control regions (ICRs) are - methylation at the paternal IGF2 loci leads to IGF2 expression) and only one allele is expressed. At the IGF2/H19 locus, IGF2 is expressed paternally (H19 on this allele is methylated and not expressed) and the maternal allele expresses H19, leaving the ICR unmethylated, allowing for the binding of a transcription factor (CTFC - zinc finger protein) that blocks IGF2's enhancer region from activating the IGF2 promoter on the maternal allele. This study showed that infants born to mothers with high Folic Acid intake, particularly of caucasian descent producing male offspring, had significantly lower H19 DMR methylation levels. This is a largely incomplete story - the mechanism is unknown, the timing of differential methylation isn't understood, whether this is clinically significant also needs to be elucidated. However, it does show: 1. imprinted sites are uniquely labile to environmental cues 2. policy recommending large folic acid intakes may not be appropriate for everyone and/or may benefit some. This also provides a potentially interesting explanation for loss of IGF2 imprinting in some Dutch famine survivors.
1. http://www.ncbi.nlm.nih.gov/pubmed/16441686
2. http://www.sciencedirect.com/science/article/pii/S1744165X08001480
3. Waterland, R. Jirtle, R. Early Nutrition, Epigenetic Changes at Transposons and Imprinted Genes, and Enhanced Susceptibility to Adult Chronic Diseases. 2004. Journal of Nutrition
4.Cooney, C. Chptr 10: Maternal Nutrition: Nutrients and Control of Expression. Nutritional Genomics: Discovering the Path To Personalized Nutrition. 2006. Wiley Press
5. http://www.ncbi.nlm.nih.gov/pubmed/9707167
6. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1001252
Quick background on genomic methylation - different CpG dinucleotides/islands/shores located throughout the genome have the potential to be methylated. This, alongside histone modifications, are responsible for cell type-specific expression patterns, X inactivation in females, silencing of transposable elements and some of the inter-individual variations in expression levels of different proteins that are not explained by coding sequences/other environmental stresses. The thinking is that the methyl groups inhibit transcription factor binding and subsequent expression rates. NOTE: tissues are extremely heterogeneous organs, filled with multiple cell types - methylation levels are expressed as a percentage of the cells within the tissue which exhibit methylation at that site- it's a caveat for anyone reading epigenetics papers to remember that until single cell RNA seq is able to be performed widely, a lot of genomic methylation studies are just looking at correlations between methylation across all cell types of that organ and overall expression - you can gain enough power from sample size and repeated measures to make that correlation seem more like causation.
Within nutritional epigenetics, folate (as well as choline and betaine) can donate methyl groups to remethylate SAM, which is responsible for methylating the DNA Methyltransferases (DNMTs). It stands to reason that at specific CpG's across the genome, a deficiency or overabundance of methyl donors may lead to differential methylation patterns between individuals - these effects can either be laid down during developmentally plastic times (gestation, early infancy/childhood) or potentially, through the simple loss of methylation over time through lack of cell maintenance - I'd like to pause for a moment here to express a thought that I had while doing some Epigenetics research at Johns Hopkins this summer - is the rat a good model for understanding nutritional epigenetics in humans? We had enough power and repeated effect to say that the availability of methyl donors most likely wasn't effecting our outcomes but I always wondered - if a dam's diet is short on methyl donors, is that deficit equally distributed across all 6-14 pups in the litter? is the deficiency only shown in a few pups? is a deficiency drastic enough to show differentially methylated regions (DMRs) even viable in rats? When looking at the pool of maternal methyl donor's direct effect on DMRs, should a single offspring species be used to draw inference for humans? Just a tangential thought I had - curious on others' opinions.
Much of nutrition's impact on epigenetics has focused on gestation and early development. The Developmental Origins of Disease Hypothesis, originally the Barker Hypothesis, argues that environment disturbances, such as low/high caloric intake through pregnancy and excess glucocorticoid exposure, can ultimately effect later disease susceptibility and pathophysiology throughout a lifetime (1) - I highly recommend reading the citation. This hypothesis largely rests upon data that shows individuals born at Low Birth Weight (LBW) are particularly susceptible to the myriad of conditions that fall under the umbrella of Metabolic Syndrome. This has interesting evolutionary implications - the placental/fetal environment may prime the infant for entering an environment of similar quality. If you are born low birth weight, your physiology (pardon my anthropomorphisizing) expects to enter a low caloric environment - however, when a LBW individual enters a world of McDonalds and soda, there is a mismatch between their expected and actual environment. A mechanism for this has been proposed via epigenetic alteration - there is evidence that excess glucocorticoids can set off a signaling cascade that alters these epigenetic modifications (2) and below, I'm going to run through a couple examples that show nutrition's impact.
The Agouti Mice - The Agouti locus is responsible for variable coat color in mice - it is expressed at specific points in development in hair follicles - a retroviral insertion into an exon of the agouti locus allows for transcription via a promotor in the long term repeat of the inserted element (intracisternal A particle) - this mutated allele allows for yellow hair pigmentation, conveying the A^VY genotype (viable yellow-phaeomelanin), compared to the wild type A^w(black-eumelanin) - this allows for an easily stratified system of classification based off of hair color. This is a simplified explanation, and there are a diverse number of strains that exhibit different coat colors. So why does a nutritional scientist care about this locus? As I mentioned before, transposable elements (like the long term repeat inserted into this locus) are methylated - these are generally viewed as being viral and need to be silenced by methylation, if they haven't already accumulated a silencing mutation. Their promoters, however, can act as alternative promoters for nearby genes (this happens with Apolipoprotein C-1). It's essential for 'normal' functioning to keep these regions methylated; this agouti region, however, has come to be included in a class of loci referred to as Metastable epialleles - basically, this means that these areas are subject to variable methylation, susceptible to environmental disturbances, and the changes in methylation state that occur are upheld mitotically - potentially, transgenerationally (3,4). Wolf (5) showed that supplementation of pregnant A^w/A^w mice's diet with methyl donors (choline, betaine, folate) and b12 led to higher % methylation of the LTR promoter, and more of an agouti color type (referred to as pseudo agouti, because the color coat is brown and not black). Dams with a supplemented diet showed an increase in mean methylation at the specific CpG sites located downstream of the insertion. Nutrition appears to be effecting genomic methylation at some loci. This example also helps to answer the side-note question that I had above: the agouti litters showed a lot of subtle variation in coat color - the availability of methyl donors seems to unequivocally effect the different pups.
The above example has a lot of limitations for human studies - mice have multiple pups, the differential methylation is only occurring at a genotype-dependent site with a retroviral insertion - to name a few. And that's why we look to Gambia...
The Gambia Study (6) used Methylation Specific Amplification Microarrays (MSAMs) to identify differentially methylated regions of the genome in peripheral blood leukocytes (PBL) and hair follicles (HF) from 8 healthy Caucasian males. Their methodology sought to identify metastable epialleles - the majority of CpG sites were found to be the same as expected, and using two tissues allowed for researchers to identify DMRs that were due to tissue-specificity. This left 107 genomic loci exhibiting inter-individual differential methylation. This number was then cut down to 40 sites after removing SNP variants and sites located in subtelomeric regions (due to copy number variation/high genetic variability - genotype has been shown to be responsible for differential methylation in past studies). They analyzed 13 of these sites from PBL and HF samples and confirmed interindividual variation within 8 sites. To confirm that these sites are variable across genetically variable populations and maintained throughout the lifetime, post-mortem tissues (liver,kidney,brain) were obtained from biking accidents in a Vietnamese population. 5 of these 8 showed inter-individual variation. The researchers went through great bioinformatic lengths to control as much for genetic variation, but as they note, it's impossible to rule out the potential that differential methylation at these loci is independent of genotype - however, by looking at three of the 5 sites in 23 monozygotic twins (drawback: they only looked at one tissue type - not able to say that the timing of when the methylation differences occurred), they were able to show that the candidate metastable epialleles weren't affected by genotype. This work allowed them to test how environmental influences, in this case maternal nutrition, altered methylation states.
Gambia is a pretty ideal population to study nutritional epigenetics. There's a strong seasonality, with a wet and a dry season, offering different food choices for pregnant mothers to consume. Rainy season infants show lower birthweights and altered susceptibility to disease. Since methyl donors affected methylation profiles in mice, the researchers predicted that seasonality of birth would affect methylation levels. They had assumed that the nutritionally-stressed rainy season would cause reduced methylation at these epialleles but instead found the opposite. Rainy season births showed over 10% increases in methylation at two of the MEs. After finding no effect on control genes, they were able to conclude that nutrition did have a unique effect on these MEs. They even went on to look at whether genetic variability would affect these results - after testing 5 of these ME's in Asian, Caucasian and Gambian individuals, quite genetically distinct populations, they found the same results. In relation to methyl donors, mothers in the rainy season had higher levels of folate - potentially explaining the higher % of methylation.
This Gambian study raises a lot of promise for nutrition's ability to effect these Metastable Epialleles. More work needs to be understood as to when the timing of differential methylation occurs, what other loci are metastable, what makes a specific site a ME, how is this extra folate interacting with SAM and DNMT's to increase methylation at just these MEs, what role are choline/betaine/serine playing in all of this? What role does dietary methyl donor availability play in maintaining Methylation at these sites?
I hope these two examples excite readers that nutrition is a key player in epigenetic plasticity and development. There are numerous other examples, particularly in colon cancer development, where folate plays a key role. The Dutch Famine is another great example of how nutrition may be effecting epigenetic states and altering disease susceptibility. The final (brief) example, as I alluded to on the first post, comes with some controversy regarding maternal folate supplementation policy:
Imprinted genes will probably get its own post at some point, but there is some evidence to show that folic acid intake may lead to aberrant methylation at imprinted loci. Imprinted genes are merely loci that exhibit mono-allelic expression - either the mother or the father's allele is methylated/unmethylated (in imprinting, methylation doesn't necessarily confer reduced expression because promoters aren't what's methylated, imprint control regions (ICRs) are - methylation at the paternal IGF2 loci leads to IGF2 expression) and only one allele is expressed. At the IGF2/H19 locus, IGF2 is expressed paternally (H19 on this allele is methylated and not expressed) and the maternal allele expresses H19, leaving the ICR unmethylated, allowing for the binding of a transcription factor (CTFC - zinc finger protein) that blocks IGF2's enhancer region from activating the IGF2 promoter on the maternal allele. This study showed that infants born to mothers with high Folic Acid intake, particularly of caucasian descent producing male offspring, had significantly lower H19 DMR methylation levels. This is a largely incomplete story - the mechanism is unknown, the timing of differential methylation isn't understood, whether this is clinically significant also needs to be elucidated. However, it does show: 1. imprinted sites are uniquely labile to environmental cues 2. policy recommending large folic acid intakes may not be appropriate for everyone and/or may benefit some. This also provides a potentially interesting explanation for loss of IGF2 imprinting in some Dutch famine survivors.
1. http://www.ncbi.nlm.nih.gov/pubmed/16441686
2. http://www.sciencedirect.com/science/article/pii/S1744165X08001480
3. Waterland, R. Jirtle, R. Early Nutrition, Epigenetic Changes at Transposons and Imprinted Genes, and Enhanced Susceptibility to Adult Chronic Diseases. 2004. Journal of Nutrition
4.Cooney, C. Chptr 10: Maternal Nutrition: Nutrients and Control of Expression. Nutritional Genomics: Discovering the Path To Personalized Nutrition. 2006. Wiley Press
5. http://www.ncbi.nlm.nih.gov/pubmed/9707167
6. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1001252
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