J. Michael Salbaum

Promoter features related to tissue specificity as measured by Shannon entropy

BackgroundThe regulatory mechanisms underlying tissue specificity are a crucial part of the development and maintenance of multicellular organisms. A genome-wide analysis of promoters in the context of gene-expression patterns in tissue surveys provides a means of identifying the general principles for these mechanisms.ResultsWe introduce a definition of tissue specificity based on Shannon entropy to rank human genes according to their overall tissue specificity and by their specificity to particular tissues. We apply our definition to microarray-based and expressed sequence tag (EST)-based expression data for human genes and use similar data for mouse genes to validate our results. We show that most genes show statistically significant tissue-dependent variations in expression level. We find that the most tissue-specific genes typically have a TATA box, no CpG island, and often code for extracellular proteins. As expected, CpG islands are found in most of the least tissue-specific genes, which often code for proteins located in the nucleus or mitochondrion. The class of genes with no CpG island or TATA box are the most common mid-specificity genes and commonly code for proteins located in a membrane. Sp1 was found to be a weak indicator of less-specific expression. YY1 binding sites, either as initiators or as downstream sites, were strongly associated with the least-specific genes.ConclusionsWe have begun to understand the components of promoters that distinguish tissue-specific from ubiquitous genes, to identify associations that can predict the broad class of gene expression from sequence data alone.

Obese-type Gut Microbiota Induce Neurobehavioral Changes in the Absence of Obesity

BACKGROUND The prevalence of mental illness, particularly depression and dementia, is increased by obesity. Here, we test the hypothesis that obesity-associated changes in gut microbiota are intrinsically able to impair neurocognitive behavior in mice. METHODS Conventionally housed, nonobese, adult male C57BL/6 mice maintained on a normal chow diet were subjected to a microbiome depletion/transplantation paradigm using microbiota isolated from donors on either a high-fat diet (HFD) or control diet. Following re-colonization, mice were subjected to comprehensive behavioral and biochemical analyses. RESULTS The mice given HFD microbiota had significant and selective disruptions in exploratory, cognitive, and stereotypical behavior compared with mice with control diet microbiota in the absence of significant differences in body weight. Sequencing-based phylogenetic analysis confirmed the presence of distinct core microbiota between groups, with alterations in α- and β-diversity, modulation in taxonomic distribution, and statistically significant alterations to metabolically active taxa. HFD microbiota also disrupted markers of intestinal barrier function, increased circulating endotoxin, and increased lymphocyte expression of ionized calcium-binding adapter molecule 1, toll-like receptor 2, and toll-like receptor 4. Finally, evaluation of brain homogenates revealed that HFD-shaped microbiota increased neuroinflammation and disrupted cerebrovascular homeostasis. CONCLUSIONS Collectively, these data reinforce the link between gut dysbiosis and neurologic dysfunction and suggest that dietary and/or pharmacologic manipulation of gut microbiota could attenuate the neurologic complications of obesity.

Maternal diabetes alters transcriptional programs in the developing embryo

BackgroundMaternal diabetes is a well-known risk factor for birth defects, such as heart defects and neural tube defects. The causative molecular mechanisms in the developing embryo are currently unknown, and the pathogenesis of developmental abnormalities during diabetic pregnancy is not well understood. We hypothesized that the developmental defects are due to alterations in critical developmental pathways, possibly as a result of altered gene expression. We here report results from gene expression profiling of exposed embryos from a mouse diabetes model.ResultsIn comparison to normal embryos at mid-gestation, we find significantly altered gene expression levels in diabetes-exposed embryos. Independent validation of altered expression was obtained by quantitative Real Time Polymerase Chain Reaction. Sequence motifs in the promoters of diabetes-affected genes suggest potential binding of transcription factors that are involved in responses to oxidative stress and/or to hypoxia, two conditions known to be associated with diabetic pregnancies. Functional annotation shows that a sixth of the de-regulated genes have known developmental phenotypes in mouse mutants. Over 30% of the genes we have identified encode transcription factors and chromatin modifying proteins or components of signaling pathways that impinge on transcription.ConclusionExposure to maternal diabetes during pregnancy alters transcriptional profiles in the developing embryo. The enrichment, within the set of de-regulated genes, of those encoding transcriptional regulatory molecules provides support for the hypothesis that maternal diabetes affects specific developmental programs.

Neural tube defect genes and maternal diabetes during pregnancy

BACKGROUND Maternal diabetes during pregnancy is a well-known teratogen that increases the risk for birth defects, such as neural tube defects (NTDs). We have previously shown that maternal diabetes profoundly affects gene expression in the developing embryo, in particular a suite of known NTD genes. In rodent experimental systems, NTDs present as phenotypes of incomplete penetrance in diabetic pregnancies. This property is difficult to reconcile with observations of consistently altered gene expression in exposed embryos. We here show that maternal diabetes increases the overall variability of gene expression levels in embryos. RESULTS Altered gene expression and increased variability of gene expression together may constitute the molecular correlates for incomplete phenotype penetrance. DISCUSSION Based on this model, we suggest that maternal diabetes reduces the precision of gene regulation in exposed individuals. Loss of precision in embryonic gene regulation may include changes to the epigenome via deregulated expression of chromatin-modifying factors. Unraveling the mechanisms underlying such epigenetic modifications in diabetic pregnancies will help to understand how teratogenic insults compromise embryonic development and possibly provide avenues for therapeutic intervention.

Diabetic embryopathy: A role for the epigenome?

Embryonic development under adverse conditions, such as maternal diabetes or obesity during pregnancy, constitutes a major risk factor for birth defects, as well as for long-term health consequences and disease susceptibility in the offspring. While contributions from epigenetic changes have been invoked previously to explain the long-term changes in terms of developmental programming, we here review how maternal metabolism may directly affect the embryonic epigenome in relationship to teratogenic processes. We consider four epigenetic modalities--DNA methylation, non-coding RNA, transcription factors, and histone modifications--and their contribution to epigenetic memory, and discuss how epigenomic changes may mediate the altered control of embryonic gene expression brought about by maternal diabetes. In combination, the epigenomic modalities serve to define transcription-permissive domains of the genome, resulting in distinct epigenomic landscapes in different developmental cell types. We evaluate experimental approaches to characterize the epigenome in adverse pregnancy conditions, highlighting the role of next-generation sequencing on the technological side, while emphasizing the necessity to study defined cell populations in terms of biologic impact. Finally, we outline the challenges in moving from findings that correlate epigenomics to developmental phenotypes to scenarios that establish teratogenic causality.

Genetic and Epigenomic Footprints of Folate

Dietary micronutrient composition has long been recognized as a determining factor for human health. Historically, biochemical research has successfully unraveled how vitamins serve as essential cofactors for enzymatic reactions in the biochemical machinery of the cell. Folate, also known as vitamin B9, follows this paradigm as well. Folate deficiency is linked to adverse health conditions, and dietary supplementation with folate has proven highly beneficial in the prevention of neural tube defects. With its function in single-carbon metabolism, folate levels affect nucleotide synthesis, with implications for cell proliferation, DNA repair, and genomic stability. Furthermore, by providing the single-carbon moiety in the synthesis pathway for S-adenosylmethionine, the main methyl donor in the cell, folate also impacts methylation reactions. It is this capacity that extends the reach of folate functions into the realm of epigenetics and gene regulation. Methylation reactions play a major role for several modalities of the epigenome. The specific methylation status of histones, noncoding RNAs, transcription factors, or DNA represents a significant determinant for the transcriptional output of a cell. Proper folate status is therefore necessary for a broad range of biological functions that go beyond the biochemistry of folate. In this review, we examine evolutionary, genetic, and epigenomic footprints of folate and the implications for human health.

Wnt signaling in caudal dysgenesis and diabetic embryopathy.

BACKGROUND Congenital defects are a major complication of diabetic pregnancy, and the leading cause of infant death in the first year of life. Caudal dysgenesis, occurring up to 200-fold more frequently in children born to diabetic mothers, is a hallmark of diabetic pregnancy. Given that there is also an at least threefold higher risk for heart defects and NTDs, it is important to identify the underlying molecular mechanisms for aberrant embryonic development. METHODS We have investigated gene expression in a transgenic mouse model of caudal dysgenesis, and in a pharmacological model using situ hybridization and quantitative real-time PCR. RESULTS We identified altered expression of several molecules that control developmental processes and embryonic growth. CONCLUSIONS The results from our models point towards major implication of altered Wnt signaling in the pathogenesis of developmental anomalies associated with embryonic exposure to maternal diabetes.

Responses of the embryonic epigenome to maternal diabetes.

Maternal diabetes and obesity are independent risk factors for neural tube defects, although it is unclear whether the effects are mediated by common pathogenic mechanisms. In this manuscript, we report a genome-wide survey of histone acetylation in neurulation stage embryos from mouse pregnancies with different metabolic conditions: maternal diabetes, and maternal consumption of a high fat content diet. We find that maternal diabetes, and independently, exposure to high-fat diet, are associated with increases and decreases of H3 and H4 histone acetylation in the embryo. Intriguingly, changes of H3K27 acetylation marks are significantly enriched near genes known to cause neural tube defects in mouse mutants. These data suggest that epigenetic changes in response to diet and metabolic condition may contribute to increased risk for neural tube defects in diabetic and obese pregnancies. Importantly, the responses to high-fat diet and maternal diabetes were distinct, suggesting that perturbed embryonic development under these conditions is mediated by different molecular pathways. This conclusion is supported by morphometric analyses that reveal a trend for maternal diabetes to delay embryonic development in the C57BL/6 strain, while high-fat diet appears to be associated with accelerated development. Taken together, our results link changes in histone acetylation to metabolic conditions during pregnancy, and implicate distinct epigenetic mechanisms in susceptibility to neural tube defects under conditions of maternal diabetes and obesity.

Identification of regulatory elements in the Isl1 gene locus

Isl1 is a LIM/homeodomain transcription factor with critical roles for the development of the heart, the nervous system and the pancreas. Both deficiency and mis-expression of Isl1 cause profound developmental defects, demonstrating the importance of proper regulation of Isl1 gene expression during development. In order to understand the mechanisms that control Isl1 expression during embryogenesis and in tissue differentiation, we initiated a screen for gene regulatory elements in the Isl1 locus using a novel dual reporter gene vector that allows screens of large genomic regions through reporter gene assays in vitro and in vivo. We identified regions from the Isl1 gene locus that confer transcriptional activity in pancreatic cell lines in vitro. Using transgenic mice, we furthermore discovered an enhancer with in vivo specificity for the developing heart, as well as visceral and posterior mesoderm. Our findings further suggest that Foxo1 as well as Gata4 contribute to the activity of this enhancer in the developing embryo. We conclude that Isl1 gene expression is controlled in modular fashion by several elements with distinct functionality. Embryonic Isl1 expression in several tissues of mesodermal origin is driven by a specific enhancer that is located 3-6kb downstream of the gene.

Maternal diet modulates the risk for neural tube defects in a mouse model of diabetic pregnancy

Pregnancies complicated by maternal diabetes have long been known to carry a higher risk for congenital malformations, such as neural tube defects. Using the FVB inbred mouse strain and the Streptozotocin-induced diabetes model, we tested whether the incidence of neural tube defects in diabetic pregnancies can be modulated by maternal diet. In a comparison of two commercial mouse diets, which are considered nutritionally replete, we found that maternal consumption of the unfavorable diet was associated with a more than 3-fold higher rate of neural tube defects. Our results demonstrate that maternal diet can act as a modifier of the risk for abnormal development in high-risk pregnancies, and provide support for the possibility that neural tube defects in human diabetic pregnancies might be preventable by optimized maternal nutrition.