Highlights

Nutritional Influences on Epigenetics and Age-Related Disease

November 4, 2011

Even with the widespread use of techniques like Genome Wide Association Studies (GWAS), there is a lot that can’t be explained by genetics alone, so the focus of many researchers today has shifted towards epigenetics and environmental influences for answers. In a recent review, researchers from Tufts University in Boston provide an overview on how environmental influences, specifically nutrition, affect epigenetics and age-related diseases.

Nutrition is a major environmental exposure, influencing almost every aspect of health and longevity. The nutrients one consumes can alter gene expression and, therefore, phenotypes. One mechanism for this is epigenetics. Epigenetic changes are heritable and modifiable marks that regulate gene transcription without altering the underlying DNA sequence and can be tweaked by nutrition in a few ways:

  • First, nutrients can provide methyl groups or act as co-enzymes for DNA and histone methylation.
  • Nutrients or bioactive ingredients can target enzymes that catalyze DNA methylation and histone modifications.
  • Finally, diet is the main input that adjusts systemic metabolism, controlling the entire cellular context leading to an organism’s epigenetic patterns.

Even though it’s been established that epigenetic profiles can be modified by nutrients, their role in physiology and disease isn’t yet thoroughly understood.

Nutrition and DNA Methylation
DNA methylation involves the addition of a methyl group at cytosine–guanine dinucleotides (CpGs). These reactions regulate the transfer of the methyl group into biological methylation reactions, and varying the intake of the components involved can alter DNA methylation. Also, many bioactive food components can alter epigenetic patterns both directly and indirectly by regulating the placement of epigenetic marks.

  • Deficiencies in nutrients like folate, B vitamins (including B-12), choline, methionine, and retinoic acid that act as methyl donors can lead to a decrease in DNA methylation.
  • Vitamin D can modify gene-specific DNA methylation.
  • Genistein can alter DNA methylation in tissue-, gene-, and life cycle–specific ways.
  • Epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, can have anti-cancer effects.
  • Apigenin (from parsley), curcumin (from turmeric), lycopene (from tomato), and sulforaphane (from cruciferous vegetables) can have inhibitory effects on DNA methyltransferases (DNMTs).

 More research is needed to determine the proper intake levels for each of these nutrients to regulate a healthy epigenome.

Nutrition and Histone Modifications
Histone proteins help pack DNA within the nucleus of a cell. Posttranslational modifications to histones function to regulate gene expression and interact with DNA methylation. Aberrant histone acetylation patterns have been linked to many diseases, including cancer, cardiac hypertrophy, and asthma. Histone acetylation is most often studied to evaluate the epigenetic effects of nutrients, bioactive components, and aging, and is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Here are a few ways nutrition can impact histones:

  • Butyrate from the digestion of dietary fiber can increase global histone acetylation in cancer cell lines.
  • Sulforaphane in broccoli, broccoli sprouts, and cabbage, as well as allyl compounds (diallyl disulfide and S-allyl mercaptocysteine) in garlic, can act as HDAC inhibitors or as possible chemopreventive agents.
  • Curcumin, found in turmeric, has been attributed with anti-inflammatory, antioxidant, and anticancer properties and has inhibitory effects on HDACs and HATs.

Aging and Nutritional Epigenetics
Nutrition influences physiology for the entire life cycle, with certain phases more sensitive to nutritional inputs than others. Recent studies suggest that nutrition in early life creates differential disease susceptibilities later on, through epigenetic mechanisms. In addition, the majority of age-related chronic diseases have a substantial lifestyle component, meaning epigenetics plays a critical role in their development.

  • Dietary folate supplementation increases both genomic and p16 promoter DNA methylation in older mice but not in young ones, demonstrating that diet may modify DNA methylation in an age-dependent way.
  • Sirtuin 1 (SIRT1) is known to deacetylate histones and nonhistone proteins, therefore regulating metabolism, stress, cellular survival, aging, inflammation, immune function, and circadian rhythms. Research shows that naturally occurring dietary polyphenols like resveratrol, curcumin, quercetin, and catechins can activate SIRT1 and may be a promising strategy against chronic inflammation, and many other age-related diseases.
  • Maternal folic acid supplementation can protect against colorectal cancer risk in children. However, the same research found that high intrauterine levels of folic acid may increase the risk of mammary tumors in the offspring, indicating the tissue specificity of epigenetic regulation.
  • Butyrate, a known HDAC inhibitor, can improve memory function in an Alzheimer’s disease mouse model when administered at an advanced stage of disease.
  • Sulforaphane and curcumin can attack cancer cells by modifying histone acetylation. Sulforaphane inhibits HDAC activity and affects cell proliferation in cancer cells. Sulforaphane and curcumin, or other bioactive components with histone modification properties, may one day be developed as cancer chemotherapeutics.
  • Exposure to high-glucose conditions can reduce H3K9 methylation at the NF-kB-p65 gene promoter and increase H3K4 methylation marks and gene expression of NF-kB targets, proving the epigenetics involved in hyperglycemia-induced vascular inflammation.
  • A high-fat diet and hypercholesterolemia shift epigenetic patterns in multiple tissues, which can result in the development of chronic disease with age.

Conclusion and future perspectives
Nutritional epigenetics is exploring gene–diet interactions, and the role nutrition and lifestyle can play in determining phenotype from genotype. Aging can also be associated with substantial changes in epigenetic patterns and age-related diseases. Some research evidence suggests certain nutrients may slow down those changes and delay disease onset, but it’s still too early to make those claims. Talented researchers around the world aim to get a better handle on a healthy epigenetic pattern of aging and find nutritional ways to maintain it, but first they’ll need to better understand:

  • The tissue-specific effects of nutrients
  • Specific developmental timing of nutritional exposures
  • If epigenetic patterns altered by nutrition and aging are programmatic or stochastic

Check out the full details yourself at Proceedings of the Nutrition Society, November 2011.

Epigenetic Aberrations in Cancer: A Prelude or Conclusion?

July 30, 2011

Do genetic mutations cause cancer, with epigenetic changes following suit, or is the process of cellular transformation kicked off by epigenetic dysregulation, with mutations ensuing therefrom? For years researchers have viewed cancer as a genetic disease, but as new epigenetic datasets continue to pour out of labs worldwide, the textbooks might need some more updating.

In a recent review, Flora Chik and her colleagues at the Department of Pharmacology and Therapeutics at McGill University's Faculty of Medicine take a look at some of latest evidence pinning epigenetics in both the initiation and progression of cancer.

Epigenetics in Cancer Initiation

In the past, cancer has been closely tied to genetic mutations. These mutations impact various regulatory cascades, ultimately enabling cells to thrive rather than die, but there are exceptions.

Some tumors, like malignant rhabdoid tumors (MRTs), are often found with an inactivated Snf5 tumor suppressor gene, the protein product of which is part of chromatin remodeling complexes. Unlike other tumors, MRT tumors display healthy genomes, providing evidence that an aberrant epigenome can lead to cancers in cells with intact genomes.

More evidence supporting epigenetic-driven cancer is provided in colon cancers, where global hypomethylation and regional promoter hypermethylation have been found in pre-cancerous regions and benign polyps before they turned malignant.

Additional studies in breast cancer have found that even some nearby normal tissue surrounding tumors have the same pattern of global hypomethylation and selective regional hypermethylation found in malignant tumors.

Other studies have shown elevated mutation rates as the result of global hypomethylation.

Such observations suggest that epigenetic changes such as methylation loss may occur before genetic instability and transformation, rather than as a consequence.


Epigenetics in Metastasis

In the review, Chik highlights that epigenetic mechanisms could play a role after initiation as well. Tumor cells of epithelial origin such as breast and prostate will often change their cytoskeletal components, lose their polarity, and become insensitive to the hormones that once regulated them, in a process called epithelial-to-mesenchymal transition (EMT).

Some of the genes commonly affected during EMT include those with CpG islands in their promoters, meaning they can be regulated epigenetically.

Among the silenced genes are those coding for the adhesion molecule E-cadherin, and for an antagonist of the WNT signaling pathway implicated in tumor growth and invasion. There are also several mesenchymal-specific genes that are turned on during EMT which contain large CpG islands found to be more demethylated in more aggressive tumors.

For nascent cancer cells to break free from their surroundings and invade new territory, they typically need to degrade the extracellular matrix (ECM). The ECM protease uPA, which can initiate a cascade resulting in proteolysis of a series of ECM proteins, has been implicated in promoting tumor invasion.

In most normal tissue, uPA is nearly undetectable, and the uPA promoter contains a large CpG island which is methylated. Studies have found uPA to be methylated in non-invasive tumor cell lines, but fully demethylated in more aggressive lines.

Setting Cancer Straight

Epigenetic modifications, unlike genetic ones, can be reversed. Opposing forces like methylation and demethylation, and their ramifications and mechanisms, are receiving increasing scrutiny in cancer research. Tools to alter the balance have even moved from laboratory investigations to clinical trials and treatments.

5-azaCdR, for example, is FDA-approved for use as a demethylating agent. During DNA replication the cytidine analog is incorporated into newly formed dsDNA. It forms covalent bonds with and sequesters DNMTs. The problem is that 5-azaCdR works non-specifically on the whole DNMT family, leading to a global loss of methylation. This has led to some unwelcome consequences, such as increased cell invasion and metastasis, presumably due to an increase in expression of various oncogenes.

As the field progresses, it will need to develop solutions like more targeted DNMT inhibitors and more specific methylating agents, and/or work with combinations of agents that in concert can find just the right balance.

Read the full review in Advances in Experimental Medicine and Biology, 2011.  

RNA-Seq Uncovers Long Non-coding RNAs in Neurogenesis and Psychiatric Disorders

September 30, 2011

In a recent study using next-generation RNA sequencing (RNA-Seq), scientists have identified long non-coding RNAs (lncRNAs) that can cause problems during neurogenesis and that sometimes lead to neuropsychiatric disorders like schizophrenia (SZ), bipolar disorder (BD), or autism spectrum disorder (ASD).

Since problems in neurogenesis include abnormal DNA methylation, transcription factors, and chromatin modifiers, researchers from the Albert Einstein College of Medicine in New York thought that using RNA-Seq on differentiating neurons would be the ideal platform to study neurogenesis. 

Neurons generated from induced pluripotent stem cells (iPSCs) were used for the project, and a wide range of changes in expression of coding genes, pseudogenes, and splice isoforms were seen, but the group was most interested in what they saw happening with lncRNAs, including:

  • lncRNAs mapping to the HOXA and HOXB gene loci were expressed in early neurogenesis
  • HOXAIRM1 is expressed in fetal brain and could be analogous to the effect of HOTAIRM1 on HOXA
  • Expression of lncRNAs Rp11-357H14.12 and AC036222.1 increased in neurogenesis and may regulate HOX genes
  • Some genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) were linked to enhanced expression of certain lncRNAs during differentiation

 

The research team's guess is that the role of lncRNAs in regulating neurogenesis is a good place for further study into where neuropsychiatric disorders get their start.

Get the full set of details at PloS ONE, September 2011.

For Research Use Only.  Not intended for human or animal therapeutic or diagnostic use.

Stem Cells Stay Poised via Epigenetics

September 12, 2011

Just like a beauty pageant contestant, or a pro athlete at crunch time, stem cells must always be poised and ready for anything. In order for embryonic stem cells to differentiate into almost any cell type, they need to keep their genetic options open, yet be ready to commit to a particular cell lineage at a moment’s notice. That’s why scientists think that epigenetic mechanisms are uniquely suited for keeping stem cells “poised” under pressure. Recently, in Stem Cell Reviews and Reports, Dr. Lyle Armstrong reviewed some of the key ways histone modifications and DNA methylation help pluripotent embryonic stem cells stay poised until it is time to commit.

Stem Cell Flexibility

Unlike mutations in a DNA sequence that can permanently alter a gene’s expression or activity, epigenetic changes are reversible, allowing cells to fine-tune gene expression in response to changing conditions. By removing an acetyl group from DNA-packing histones or by adding a methyl group to a cytosine in DNA, these cells have the means to dial in which genes are expressed and when.

This flexibility is very important for stem cells, which suppress lineage-specific genes until they receive the signal to differentiate into a particular cell type. Then they must quickly and selectively activate genes important for that differentiation state, while silencing pluripotency genes and genes specific to other cell types.

Ambivalent Chromatin

In 2006 researchers made the puzzling discovery that in embryonic stem cells, the promoters of many genes important for differentiation contain both activating and silencing epigenetic marks. Large stretches of DNA were found to contain histone proteins modified with both the repressive H3K27me modification (indicating a methyl group on lysine 27 of the histone protein H3) and the activating H3K4me modification (methyl group on lysine 4 of histone H3). Until then, researchers had though that the functions of these two histone modifications were mutually exclusive.

It’s not that stem cells are sending mixed signals. Research indicates this so-called “bivalent” state poises cells to quickly respond to differentiation signals. Bivalent domains frequently occur in differentiation-associated transcription factor genes that are expressed at very low levels in embryonic stem cells. When the cell needs to differentiate, so the hypothesis goes, it can quickly remove the silencing H3K27me modification and be “transcription-ready” without waiting for histone-modifying enzymes to add an activating H3K4me modification.

DNA Methylation in the Mix

In addition to histone modifications, cells make use of reversible DNA methylation to silence or activate genes. DNA methyltransferases can add a methyl group to the C5 position of cytosine, usually in the context of CpG dinucleotides. When clusters of CpGs in a promoter, known as CpG islands, become heavily methylated, the associated gene is often silenced. Recently researchers have discovered other modified forms of cytosine, such as 5-hydroxymethylcytosine and 5-formylcytosine, that may also influence gene expression.

Overall, stem cells show less DNA methylation than somatic cells do, although tissue-specific genes are often highly methylated and therefore silenced. In genes with H3K4me histone modifications, DNA methylation is greatly reduced, suggesting interplay between the histone modification and DNA methylation systems.

Differentiation Changes Everything

When stem cells differentiate, their chromatin converts from a loose, open architecture to a more compact, restrictive conformation. Histones become less dynamic and remain bound to DNA for longer periods of time. This closed chromatin state limits the range of genes that the cell can express, probably by blocking access of transcription factors and other proteins to the DNA.

Histone modifications change as differentiation progresses. An early event is phosphorylation of serine 10 on histone H3 coupled to acetylation of H3 lysine 14, which may pave the way for other chromatin modifications.

The bivalent state of histone modifications typically resolves during differentiation. Genes that undergo permanent repression in the differentiated state lose the H3K4 methylation mark while retaining H3K27 methylation. Subsequent H3K9 methylation further establishes a highly repressive chromatin state. When cells lose H3K4 methylation, they begin to methylate DNA in those regions. These changes shut down the expression of pluripotency-associated genes and genes specific for other cell lineages.

Meanwhile, genes required for the target lineage get activated. This is where the “poised” epigenetic state becomes important, because cells can now quickly activate lineage-specific genes. Cells remove the repressive H3K27 modification, opening up the chromatin structure for further histone methylation and acetylation. When the chromatin structure is sufficiently open, transcription factors can bind to DNA and activate the transcription of lineage-specific genes.

Decoding Epigenetics in Differentiation

Understanding the complex cascade of epigenetic events of differentiation is obviously important if scientists wish to fully harness the awesome potential of embryonic stem cells to regenerate any type of cell in the body. Although researchers have identified specific chromatin modifications important for this process, the precise timing and full spectrum of modifications remain to be elucidated.

More complete knowledge of these modifications will also allow researchers to compare the differentiation of embryonic stem cells with that of induced pluripotent stem (iPS) cells. If iPS cells are to be used safely and effectively in regenerative medicine, scientists need to ensure that their differentiation closely mimics that of natural embryonic stem cells.

To learn more about the incredible poise and personality of embryonic stem cells, check out this great review in Stem Cell Reviews and Reports, August 2011.

miRNA SNPs and Cancer: What a Difference a Base Makes

July 15, 2011

In the past decade, scientists have realized that small, non–protein-coding RNAs can substantially alter gene expression. MicroRNAs (miRNAs) block the translation of their mRNA targets, usually by binding to complementary sequences in the mRNA's 3'-untranslated region. Increasing evidence suggests that this powerful form of posttranscriptional gene regulation goes awry in cancer, contributing to the disease. By characterizing the more than 1,000 miRNA sequences thought to reside in the human genome, researchers hope to identify genetic variants known as single-nucleotide polymorphisms (SNPs) that could someday be used to potentially diagnose and treat cancer.

SNPs Make Their Point in miRNA Regulation

It's now widely recognized that point mutations in protein oncogenes and tumor suppressors can cause cancer. Arguably, single-base changes in miRNAs have an even greater potential to throw a monkey wrench into the cellular works. For one, miRNAs' small size–20 to 22 nucleotides in length–makes them particularly vulnerable to the effects of single-nucleotide changes. A point mutation in the 2- to 7-nucleotide miRNA seed region, which is crucial for base pairing to the mRNA target, can abolish binding to specific mRNAs or create new targets. Mutations elsewhere in the miRNA can affect its structure, expression level, or processing from a larger miRNA precursor.

Another reason why small differences in miRNA sequences can drastically impact cells is that a single miRNA can have multiple mRNA targets. For example, when researchers transfected human cells with either miR-1 or miR-124, each miRNA downregulated the RNA expression levels of about 100 genes (Nature 2005, 433:769–773). Therefore, a single miRNA point mutation could result in widespread gene deregulation.

In addition to point mutations in the miRNA sequences themselves, single-nucleotide changes in the miRNA's binding site on target transcripts could prevent translational inhibition, leading to protein overexpression. Moreover, point mutations in proteins involved in miRNA processing, such as Drosha and Dicer, could affect the levels of functional miRNAs.

Human cancers express abnormal levels of miRNAs. To find out why, researchers have tried to identify differences in miRNA-related sequences between normal and tumor cells. "We thought that maybe miRNAs become mutated in tumor cells", explains Joanne Weidhaas, an oncologist and researcher at Yale University School of Medicine. Surprisingly, however, Weidhaas and others found that such tumor-acquired miRNA point mutations are exceedingly rare.

Yet when researchers compared miRNA sequences and targets in cancer patients with those of healthy volunteers, they discovered that cancer patients had a higher incidence of certain sequence variants, or SNPs, in miRNA-related genes. "We found differences in miRNA target sequences that people are born with, or germline SNPs", says Weidhaas. She notes that, in general, the term SNP refers to silent single-nucleotide differences in DNA sequences among individuals. In contrast, miRNA SNPs are much more likely to have functional consequences.

miRNA-Related SNPs and Cancer

Indeed, some miRNA-related SNPs, possibly in combination with other genetic or environmental factors, increase a person's risk of developing cancer. For example, researchers linked a germline SNP within the miR-16-1 gene to familial chronic lymphocytic leukemia (N. Engl. J. Med. 2005, 353:1793–1801). The SNP, which involves a C-to-T substitution in the miR-16-1 precursor, causes reduced levels of mature miR-16-1.

Weidhaas and her colleagues discovered that a T-to-G SNP in the 3'-untranslated region of the KRAS oncogene increased the risk for non-small cell lung cancer (Cancer Res. 2008, 68:8535–8540) and ovarian cancer (Cancer Res. 2010, 70:509–6515). This SNP lies within thebinding site for the let-7 miRNA, and results in boosted levels of KRAS in vitro.

Mira Dx, a company cofounded by Weidhaas, markets a diagnostic test for the KRAS 3'-untranslated region variant. Because 25% of women with ovarian cancer express the KRAS variant (compared with 6–10% of the general population), the test can help diagnose the disease, as well as inform female family members who also carry the KRAS variant of their increased risk. Weidhaas says that diagnostic tests based on miRNA gene sequences are more straightforward and sensitive than those that rely on the detection of protein levels, which can fluctuate under different physiological or storage conditions.

miRNA-related SNPs can influence not only a person's risk of developing cancer, but also their prognosis and response to treatment. Patients with non-small cell lung cancer who had a particular miR-196a2 SNP showed reduced survival (J. Clin. Invest. 2008, 118:2600–2608). A SNP in the 3'-untranslated region of the dihydrofolate reductase gene conferred resistance to the chemotherapy drug methotrexate by inhibiting the binding of miR-24 (Proc. Natl. Acad. Sci. USA 2007, 104:13513–13518).

The Next Biomarker?

New discoveries open the door for individualized cancer treatments that target each patient's particular miRNA-related SNP. “It's a whole new avenue of drug development,” says Weidhaas. But first, researchers need to perform clinical studies to confirm the findings in large, well-characterized populations. In addition, they need to elucidate just how miRNA SNPs influence response to radiation and chemotherapies.

Although it's likely that researchers have only scratched the surface of miRNA-related SNPs' effects on cancer, Weidhaas believes that these small but powerful miRNA changes have the potential to revolutionize our understanding and treatment of cancer. "It's a changed paradigm", she says. "The old thinking was that mutations had to screw up a protein or they can't matter. Now evidence is building that miRNA-related SNPs can significantly impact a person's risk of cancer and their tumor biology."


July 15, 2011