Transcriptome-wide analysis of changes in the fetal placenta associated with prenatal arsenic exposure in the New Hampshire Birth Cohort Study.

BACKGROUND: Increasing evidence suggests that prenatal exposure to arsenic, even at common environmental levels, adversely affects child health. These adverse effects include impaired fetal growth, which can carry serious health implications lifelong. However, the mechanisms by which arsenic affects fetal health and development remain unclear. METHODS: We addressed this question using a group of 46 pregnant women selected from the New Hampshire Birth Cohort Study (NHBCS), a US cohort exposed to low-to-moderate arsenic levels in drinking water through the use of unregulated private wells. Prenatal arsenic exposure was assessed using maternal urine samples taken at mid-gestation. Samples of the fetal portion of the placenta were taken from the base of the umbilical cord insertion at the time of delivery, stored in RNAlater and frozen. We used RNA sequencing to analyze changes in global gene expression in the fetal placenta associated with in utero arsenic exposure, adjusting for maternal age. Gene set enrichment analysis and enrichment mapping were then used to identify biological processes represented by the differentially expressed genes. Since our previous analyses have identified considerable sex differences in placental gene expression associated with arsenic exposure, we analyzed male and female samples separately. RESULTS: At FDR < 0.05, no genes were differentially expressed in female placenta, while 606 genes were differentially expressed in males. Genes showing the most significant associations with arsenic exposure in females were LEMD1 and UPK3B (fold changes 2.51 and 2.48), and in males, FIBIN and RANBP3L (fold changes 0.14 and 0.15). In gene set enrichment analyses, at FDR < 0.05, a total of 211 gene sets were enriched with differentially expressed genes in female placenta, and 154 in male placenta. In female but not male placenta, 103 of these gene sets were also associated with reduced birth weight. CONCLUSIONS: Our results reveal multiple biological functions in the fetal placenta that are potentially affected by increased arsenic exposure, a subset of which is sex-dependent. Further, our data suggest that in female infants, the mechanisms underlying the arsenic-induced reduction of birth weight may involve activation of stress response pathways.

Prenatal arsenic exposure alters the placental expression of multiple epigenetic regulators in a sex-dependent manner.

BACKGROUND: Prenatal exposure to arsenic has been linked to a range of adverse health conditions in later life. Such fetal origins of disease are frequently the result of environmental effects on the epigenome, leading to long-term alterations in gene expression. Several studies have demonstrated effects of prenatal arsenic exposure on DNA methylation; however the impact of arsenic on the generation and decoding of post-translational histone modifications (PTHMs) is less well characterized, and has not been studied in the context of prenatal human exposures. METHODS: In the current study, we examined the effect of exposure to low-to-moderate levels of arsenic in a US birth cohort, on the expression of 138 genes encoding key epigenetic regulators in the fetal portion of the placenta. Our candidate genes included readers, writers and erasers of PTHMs, and chromatin remodelers. RESULTS: Arsenic exposure was associated with the expression of 27 of the 138 epigenetic genes analyzed. When the cohort was stratified by fetal sex, arsenic exposure was associated with the expression of 40 genes in male fetal placenta, and only 3 non-overlapping genes in female fetal placenta. In particular, we identified an inverse relationship between arsenic exposure and expression of the gene encoding the histone methyltransferase, PRDM6 (p < 0.001). Mutation of PRDM6 has been linked to the congenital heart defect, patent ductus arteriosus. CONCLUSIONS: Our findings suggest that prenatal arsenic exposure may have sex-specific effects on the fetal epigenome, which could plausibly contribute to its subsequent health impacts.

The aquaglyceroporin AQP9 contributes to the sex-specific effects of in utero arsenic exposure on placental gene expression.

BACKGROUND: Sex-specific factors play a major role in human health and disease, including responses to environmental stresses such as toxicant exposure. Increasing evidence suggests that such sex differences also exist during fetal development. In a previous report using the resources of the New Hampshire Birth Cohort Study (NHBCS), we found that low-to-moderate in utero exposure to arsenic, a highly toxic and widespread pollutant, was associated with altered expression of several key developmental genes in the fetal portion of the placenta. These associations were sex-dependent, suggesting that in utero arsenic exposure differentially impacts male and female fetuses. In the present study, we investigated the molecular basis for these sex-specific responses to arsenic. METHODS: Using NanoString technology, we further analyzed the fetal placenta samples from the NHBCS for the expression of genes encoding arsenic transporters and metabolic enzymes. Multivariable linear regression analysis was used to examine their relationship with arsenic exposure and with key developmental genes, after stratification by fetal sex. RESULTS: We found that maternal arsenic exposure was strongly associated with expression of the AQP9 gene, encoding an aquaglyceroporin transporter, in female but not male fetal placenta. Moreover, AQP9 expression associated with that of a subset of female-specific arsenic-responsive genes. CONCLUSIONS: Our results suggest that AQP9 is upregulated in response to arsenic exposure in female, but not male, fetal placenta. Based on these results and prior studies, increased AQP9 expression may lead to increased arsenic transport in the female fetal placenta, which in turn may alter the expression patterns of key developmental genes that we have previously shown to be associated with arsenic exposure. Thus, this study suggests that AQP9 may play a role in the sex-specific effects of in utero arsenic exposure.

GLI3 Links Environmental Arsenic Exposure and Human Fetal Growth.

Although considerable evidence suggests that in utero arsenic exposure affects children's health, these data are mainly from areas of the world where groundwater arsenic levels far exceed the World Health Organization limit of 10 µg/L. We, and others, have found that more common levels of in utero arsenic exposure may also impact children's health. However, the underlying molecular mechanisms are poorly understood. To address this issue, we analyzed the expression of key developmental genes in fetal placenta in a birth cohort of women using unregulated water supplies in a US region with elevated groundwater arsenic. We identified several genes whose expression associated with maternal arsenic exposure in a fetal sex-specific manner. In particular, expression of the HEDGEHOG pathway component, GLI3, in female placentae was both negatively associated with arsenic exposure and positively associated with infant birth weight. This suggests that modulation of GLI3 in the fetal placenta, and perhaps in other fetal tissues, contributes to arsenic's detrimental effects on fetal growth. We showed previously that arsenic-exposed NIH3T3 cells have reduced GLI3 repressor protein. Together, these studies identify GLI3 as a key signaling node that is affected by arsenic, mediating a subset of its effects on developmental signaling and fetal health.

Epigenetic pathways and glioblastoma treatment: insights from signaling cascades.

There is an urgent need to identify novel therapies for glioblastoma (GBM) as most therapies are ineffective. A first step in this process is to identify and validate targets for therapeutic intervention. Epigenetic modulators have emerged as attractive drug targets in several cancers including GBM. These epigenetic regulators affect gene expression without changing the DNA sequence. Recent studies suggest that epigenetic regulators interact with drivers of GBM cell and stem-like cell proliferation. These drivers include components of the Notch, Hedgehog, and Wingless (WNT) pathways. We highlight recent studies connecting epigenetic and signaling pathways in GBM. We also review systems and big data approaches for identifying patient specific therapies in GBM. Collectively, these studies will identify drug combinations that may be effective in GBM and other cancers.

Abelson interactor 1 (ABI1) and its interaction with Wiskott-Aldrich syndrome protein (wasp) are critical for proper eye formation in Xenopus embryos.

Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.

Gsx transcription factors repress Iroquois gene expression.

We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.

Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis.

The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.

Complementary expression of HSPG 6-O-endosulfatases and 6-O-sulfotransferase in the hindbrain of Xenopus laevis.

Heparan sulfate proteoglycans (HSPGs) are abundant cell surface molecules, consisting of glycosaminoglycan (GAG) chains bound to a protein core. There is high diversity in the sulfation pattern within each GAG chain, creating specific binding sites for many proteins including cell signalling factors, whose activities and distribution are modified by their association with HSPGs (Danesin et al., 2006; Freeman et al., 2008). Here, we describe the distinct expression of three enzymes which modify the 6-O-sulfation state of HSPGs: two 6-O-endosulfatases (Sulf1 and Sulf2), and a 6-O-sulfotransferase (6OST-1). We use in situ hybridisation to determine the spatial distribution of transcripts during tailbud stages of Xenopus laevis development, with a particular focus on neural regions where the 6-O-sulfatases are expressed ventrally and the 6-O-sulfotransferase is restricted dorsally. The complementary expression of these enzymes in the hindbrain and neural tube suggest a role for specific HSPG structure in dorsoventral patterning, possibly through modifying the activity or distribution of signalling molecules such as BMP, Sonic hedgehog, Wnt and/or FGF.