Role of endolysosome function in iron metabolism and brain carcinogenesis.

Iron, the most abundant metal in human brain, is an essential microelement that regulates numerous cellular mechanisms. Some key physiological roles of iron include oxidative phosphorylation and ATP production, embryonic neuronal development, formation of iron-sulfur clusters, and the regulation of enzymes involved in DNA synthesis and repair. Because of its physiological and pathological importance, iron homeostasis must be tightly regulated by balancing its uptake, transport, and storage. Endosomes and lysosomes (endolysosomes) are acidic organelles known to contain readily releasable stores of various cations including iron and other metals. Increased levels of ferrous (Fe2+) iron can generate reactive oxygen species (ROS) via Fenton chemistry reactions and these increases can damage mitochondria and genomic DNA as well as promote carcinogenesis. Accumulation of iron in the brain has been linked with aging, diet, disease, and cerebral hemorrhage. Further, deregulation of brain iron metabolism has been implicated in carcinogenesis and may be a contributing factor to the increased incidence of brain tumors around the world. Here, we provide insight into mechanisms by which iron accumulation in endolysosomes is altered by pH and lysosome membrane permeabilization. Such events generate excess ROS resulting in mitochondrial DNA damage, fission, and dysfunction, as well as DNA oxidative damage in the nucleus; all of which promote carcinogenesis. A better understanding of the roles that endolysosome iron plays in carcinogenesis may help better inform the development of strategic therapeutic options for cancer treatment and prevention.

Establishing a role for environmental toxicant exposure induced epigenetic remodeling in malignant transformation.

Humans are exposed to a wide variety of environmental exposures throughout their lifespan. These include both naturally occurring toxins and chemical toxicants like pesticides, herbicides, and industrial chemicals, many of which have been implicated as possible contributors to human disease susceptibility [1-3]. We, and others, have hypothesized that environmental exposures may cause adaptive epigenetic changes in regenerative cell populations and developing organisms, leading to abnormal gene expression and increased disease susceptibility later in life [3]. Common epigenetic changes include changes in miRNA expression, covalent histone modifications, and methylation of DNA. Importantly, due to their heritable nature, abnormal epigenetic modifications which occur within stem cells may be particularly deleterious. Abnormal epigenetic changes in regenerative cell linages can be passed onto a large population of daughter cells and can persist for long periods of time. It is well established that an accumulation of epigenetic changes can lead to many human diseases including cancer [4-6]. Subsequently, it is imperative that we increase our understanding of how common environmental toxins and toxicants can induce epigenetic changes, particularly in stem cell populations. In this review, we will discuss how common environmental exposures in the United States and around the world may lead to epigenetic changes and discuss potential links to human disease, including cancer.

Environmental Exposures, the Epigenome, and African American Women's Health.

Stress is a common feature of modern life, but both the extent of exposure to stressors and the downstream effects of these stress exposures can vary considerably among individuals, communities, and populations. When individuals are exposed to repeated or chronic stress, wear and tear on the body can accumulate and manifest in many ways. The term "allostatic load" represents the physiological consequences of repeated or chronic exposure to environmental stressors and is linked to fluctuating and/or heightened neural or neuroendocrine responses. African American women are one population subgroup that has been identified as potentially having both an elevated allostatic load and an enhanced resilience to external factors. One mechanism by which environmental stressors may impact human health is via epigenetic remodeling of the genome. This review will focus on what is known about how different types of environmental stressors may affect the epigenome and explore links between epigenetic reprogramming and altered allostatic load and resilience as it pertains to African American women's health.

A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing.

Adult cancers may derive from stem or early progenitor cells. Epigenetic modulation of gene expression is essential for normal function of these early cells but is highly abnormal in cancers, which often show aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors. We find that for such genes, both normal and malignant embryonic cells generally lack the hypermethylation of DNA found in adult cancers. In embryonic stem cells, these genes are held in a 'transcription-ready' state mediated by a 'bivalent' promoter chromatin pattern consisting of the repressive mark, histone H3 methylated at Lys27 (H3K27) by Polycomb group proteins, plus the active mark, methylated H3K4. However, embryonic carcinoma cells add two key repressive marks, dimethylated H3K9 and trimethylated H3K9, both associated with DNA hypermethylation in adult cancers. We hypothesize that cell chromatin patterns and transient silencing of these important regulatory genes in stem or progenitor cells may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing during tumor initiation and progression.

The cholesterol metabolite 27-hydroxycholesterol regulates p53 activity and increases cell proliferation via MDM2 in breast cancer cells.

Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction?

Chromatin alterations have been associated with all stages of tumour formation and progression. The best characterized are epigenetically mediated transcriptional-silencing events that are associated with increases in DNA methylation - particularly at promoter regions of genes that regulate important cell functions. Recent evidence indicates that epigenetic changes might 'addict' cancer cells to altered signal-transduction pathways during the early stages of tumour development. Dependence on these pathways for cell proliferation or survival allows them to acquire genetic mutations in the same pathways, providing the cell with selective advantages that promote tumour progression. Strategies to reverse epigenetic gene silencing might therefore be useful in cancer prevention and therapy.

DNA methylation in complex disease: applications in nursing research, practice, and policy.

DNA methylation is an epigenomic modification that is essential to normal human development and biological processes. DNA methylation patterns are heritable and dynamic throughout the life span. Environmental exposures can alter DNA methylation patterns, contributing to the development of complex disease. Identification and modulation of environmental factors influencing disease susceptibility through alterations in DNA methylation are amenable to nursing intervention and form the basis for individualized patient care. Here we describe the evidence supporting the translation of DNA methylation analyses as a tool for screening, diagnosis, and treatment of complex disease in nursing research and practice. The ethical, legal, social, and economic considerations of advances in genomics are considered as a model for epigenomic policy. We conclude that contemporary and informed nurse scientists and clinicians are uniquely poised to apply innovations in epigenomic research to clinical populations and develop appropriate policies that guide equitable and ethical use of new strategies to improve patient care.

Genetic and environmental reprogramming of the sarcoma epigenome.

Sarcomas are rare and heterogenous mesenchymal tumors occurring in soft tissue and bone. The World Health Organization Classification of sarcomas comprises more than hundred different entities which are very diverse in their molecular, genetic and epigenetic signatures as they are in their clinical presentations and behaviors. While sarcomas can be associated with an underlying hereditary cancer predisposition, most sarcomas developed sporadically without identifiable cause. Sarcoma oncogenesis involves complex interactions between genetic, epigenetic and environmental factors which are intimately related and intensively studied. Several molecular discoveries have been made over the last decades leading to the development of new therapeutic avenues. Sarcoma research continues its effort toward a more specific and personalized approach to all sarcoma sub-types to improve patient outcomes and this through world-wide collaboration. This chapter on "Genetic and Environmental Reprogramming of the Sarcoma Epigenome" provides a comprehensive review of general concepts and epidemiology of sarcoma as well as a detailed description of the genetic, molecular and epigenetic alterations seen in sarcomas, their therapeutic implications and ongoing research. This review also presents evidenced-based data on the environmental and occupational factors possibly involved in the etiology of sarcomas and a brief discussion on the role of the microbiome in sarcoma.

WEE1 inhibition augments CDC7 (DDK) inhibitor-induced cell death in Ewing sarcoma by forcing premature mitotic entry and mitotic catastrophe.

Ewing sarcoma is an aggressive childhood cancer for which treatment options remain limited and toxic. There is an urgent need for the identification of novel therapeutic strategies. Our group has recently shown that Ewing cells rely on the S-phase kinase CDC7 (DDK) to maintain replication rates and cell viability and that DDK inhibition causes an increase in the phosphorylation of CDK1 and a significant delay in mitotic entry. Here, we expand on our previous findings and show that DDK inhibitor-induced mitotic entry delay is dependent upon WEE1 kinase. Specifically, WEE1 phosphorylates CDK1 and prevents mitotic entry upon DDK inhibition due to the presence of under-replicated DNA, potentially limiting the cytotoxic effects of DDK inhibition. To overcome this, we combined the inhibition of DDK with the inhibition of WEE1 and found that this results in elevated levels of premature mitotic entry, mitotic catastrophe, and apoptosis. Importantly, we have found that DDK and WEE1 inhibitors display a synergistic relationship with regards to reducing cell viability of Ewing sarcoma cells. Interestingly, the cytotoxic nature of this combination can be suppressed by the inhibition of CDK1 or microtubule polymerization, indicating that mitotic progression is required to elicit the cytotoxic effects. This is the first study to display the potential of utilizing the combined inhibition of DDK and WEE1 for the treatment of cancer. We believe this will offer a potential therapeutic strategy for the treatment of Ewing sarcoma as well as other tumor types that display sensitivity to DDK inhibitors.

CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma.

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK's diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma.

Combined BRCA2 and MAGEC3 Expression Predict Outcome in Advanced Ovarian Cancers.

Like BRCA2, MAGEC3 is an ovarian cancer predisposition gene that has been shown to have prognostic significance in ovarian cancer patients. Despite the clinical significance of each gene, no studies have been conducted to assess the clinical significance of their combined expression. We therefore sought to determine the relationship between MAGEC3 and BRCA2 expression in ovarian cancer and their association with patient characteristics and outcomes. Immunohistochemical staining was quantitated on tumor microarrays of human tumor samples obtained from 357 patients with epithelial ovarian cancer to ascertain BRCA2 expression levels. In conjunction with our previously published MAGEC3 expression data, we observed a weak inverse correlation of MAGEC3 with BRCA2 expression (r = −0.15; p < 0.05) in cases with full-length BRCA2. Patients with optimal cytoreduction, loss of MAGEC3, and detectable BRCA2 expression had better overall (median OS: 127.9 vs. 65.3 months, p = 0.035) and progression-free (median PFS: 85.3 vs. 18.8 months, p = 0.002) survival compared to patients that were BRCA2 expressors with MAGEC3 normal levels. Our results suggest that combined expression of MAGEC3 and BRCA2 serves as a better predictor of prognosis than each marker alone.

Clinicopathologic and molecular analysis of a BCOR-CCNB3 undifferentiated sarcoma of the kidney reveals significant epigenetic alterations.

Undifferentiated soft tissue sarcomas (UDSTSs) are a group of mesenchymal tumors that remain a diagnostic challenge because of their morphologic heterogeneity and unclear histologic origin (Peters et al., Mod Pathol28: 575 [2015]). In this case report, we present the first multiomics molecular signature for a BCOR-CCNB3 sarcoma (BCS) that includes mutation analysis, gene expression, DNA methylation, and micro RNA (miRNA) expression. We identify a paucity of additional mutations in this tumor and detail that there is significant dysregulation of gene expression of epigenetic remodeling agents including key members of the PRC, Sin3A/3b, NuRD, and NcoR/SMRT complexes and the DNA methyltransferases DNMT1, DNMT3a, and DNMT3b. This is accompanied by significant DNA methylation changes and dysregulation of multiple miRNAs with known links to tumorigenesis. This study significantly increases our understanding of the BCOR effects on fusion-positive undifferentiated sarcomas at both the genomic and epigenomic level and suggests that as better-tailored and more refined treatment algorithms continue to evolve, epigenetic modifying agents should be further evaluated for their efficacy against these tumors.

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.

PcG proteins, DNA methylation, and gene repression by chromatin looping.

Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream ( approximately 30 kb) of and extending approximately 60 kb around one such gene, GATA-4, is organized-in Tera-2 undifferentiated embryonic carcinoma (EC) cells-in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)-mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a approximately 60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.

Exploiting Replication Stress as a Novel Therapeutic Intervention.

Ewing sarcoma is an aggressive pediatric tumor of the bone and soft tissue. The current standard of care is radiation and chemotherapy, and patients generally lack targeted therapies. One of the defining molecular features of this tumor type is the presence of significantly elevated levels of replication stress as compared with both normal cells and many other types of cancers, but the source of this stress is poorly understood. Tumors that harbor elevated levels of replication stress rely on the replication stress and DNA damage response pathways to retain viability. Understanding the source of the replication stress in Ewing sarcoma may reveal novel therapeutic targets. Ewing sarcomagenesis is complex, and in this review, we discuss the current state of our knowledge regarding elevated replication stress and the DNA damage response in Ewing sarcoma, one contributor to the disease process. We will also describe how these pathways are being successfully targeted therapeutically in other tumor types, and discuss possible novel, evidence-based therapeutic interventions in Ewing sarcoma. We hope that this consolidation will spark investigations that uncover new therapeutic targets and lead to the development of better treatment options for patients with Ewing sarcoma. IMPLICATIONS: This review uncovers new therapeutic targets in Ewing sarcoma and highlights replication stress as an exploitable vulnerability across multiple cancers.

Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

Importance of measuring endolysosome, cytosolic, and extracellular pH in understanding the pathogenesis of and possible treatments for glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is a very aggressive form of brain cancer that carries with it a tragically poor prognosis. As with many other forms of cancer, the extracellular environment near GBM tumors is acidified and is relevant to the pathogenesis of GBM because decreased pH promotes tumor cell invasion, increases angiogenesis, decreases immune surveillance, and increases resistance to possible treatments. Recently, vacuolar ATPase (v-ATPase), a proton pump that helps maintain the acidic environment in endosomes and lysosomes (hereafter referred to endolysosomes) as well as proton gradients across the plasma membrane, was identified as a novel therapeutic target for GBM. However, information is lacking about cancer cell and tissue pH of endolysosomes, cytosol and extracellular fluid. AIM: Here, we measured endolysosome, cytosolic, and extracellular pH in U87MG cells in the absence and presence of the v-ATPase inhibitor bafilomycin A1. METHODS: In vitro measurements of U87MG cells were conducted using LysoSensor dye and a Lysosome-RFP dye for lysosome pH, BCECF-AM for cytosolic pH, and a pH-sensitive microprobe for extracellular pH. RESULTS: Bafilomycin A1 increased endolysosome pH from 5.28 to 5.57, decreased cytosolic pH from 7.01 to 6.46, and increased extracellular pH from 7.18 to 7.40. CONCLUSIONS: Here, we report the ability to make pH measurements in U87MG glioblastoma cells and discuss these results in the context of GBM pathogenesis and possible treatment. This might be of some importance in understanding the pathogenesis of GBM because the highly regulated stores of hydrogen (H+) ions in endolysosomes can influence cytosolic and extracellular pH as well as the distribution, numbers, and sizes of endolysosomes.

Role of endolysosomes and pH in the pathogenesis and treatment of glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a Grade IV astrocytoma with an aggressive disease course and a uniformly poor prognosis. Pathologically, GBM is characterized by rapid development of primary tumors, diffuse infiltration into the brain parenchyma, and robust angiogenesis. The treatment options that are limited and largely ineffective include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide. RECENT FINDINGS: Similar to many other forms of cancer, the extracellular environment near GBM tumors is acidified. Extracellular acidosis is particularly relevant to tumorgenesis and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio-treatment. Factors known to participate in the acidification process are nutrient starvation, oxidative stress, hypoxia and high levels of anaerobic glycolysis that lead to increases in lactate. Also involved are endosomes and lysosomes (hereafter termed endolysosomes), acidic organelles with highly regulated stores of hydrogen (H+) ions. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that are known to affect the number, sizes and distribution patterns of these organelles within cells. Recently, vacuolar ATPase (v-ATPase), the main proton pump that is responsible for maintaining the acidic environment in endolysosomes, was identified as a novel therapeutic target for glioblastoma. CONCLUSIONS: Thus, a greater understanding of the role of endolysosomes in regulating cellular and extracellular acidity could result in a better elucidation of GBM pathogenesis and new therapeutic strategies.

Mtgr1 is a transcriptional corepressor that is required for maintenance of the secretory cell lineage in the small intestine.

Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.

Effects of Maternal Vitamin D Supplementation on the Maternal and Infant Epigenome.

INTRODUCTION: Mothers and infants are at high risk for inadequate vitamin D status. Mechanisms by which vitamin D may affect maternal and infant DNA methylation are poorly understood. OBJECTIVE: This study quantified the effects of vitamin D3 supplementation on DNA methylation in pregnant and lactating women and their breastfed infants. MATERIALS AND METHODS: In this randomized controlled pilot study, pregnant women received vitamin D3 400 international units (IU) (n = 6; control) or 3,800 IU (n = 7; intervention) daily from late second trimester through 4-6 weeks postpartum. Epigenome-wide DNA methylation was quantified in leukocytes collected from mothers at birth and mother-infant dyads at 4-6 weeks postpartum. RESULTS: At birth, intervention group mothers showed DNA methylation gain and loss at 76 and 89 cytosine-guanine (CpG) dinucleotides, respectively, compared to controls. Postpartum, methylation gain was noted at 200 and loss at 102 CpGs. Associated gene clusters showed strongest biologic relevance for cell migration/motility and cellular membrane function at birth and cadherin signaling and immune function at postpartum. Breastfed 4-6-week-old infants of intervention mothers showed DNA methylation gain and loss in 217 and 213 CpGs, respectively, compared to controls. Genes showing differential methylation mapped most strongly to collagen metabolic processes and regulation of apoptosis. CONCLUSIONS: Maternal vitamin D supplementation during pregnancy and lactation alters DNA methylation in mothers and breastfed infants. Additional work is needed to fully elucidate the short- and long-term biologic effects of vitamin D supplementation at varying doses, which could hold important implications for establishing clinical recommendations for prenatal and offspring health promotion.