Epigenomic Landscapes of hESC-Derived Neural Rosettes: Modeling Neural Tube Formation and Diseases

We currently lack a comprehensive understanding of the mechanisms underlying neural tube formation and their contributions to neural tube defects (NTDs). Developing a model to study such a complex morphogenetic process, especially one that models human-specific aspects, is critical. Three-dimensional, human embryonic stem cell (hESC)-derived neural rosettes (NRs) provide a powerful resource for in vitro modeling of human neural tube formation. Epigenomic maps reveal enhancer elements unique to NRs relative to 2D systems. A master regulatory network illustrates that key NR properties are related to their epigenomic landscapes. We found that folate-associated DNA methylation changes were enriched within NR regulatory elements near genes involved in neural tube formation and metabolism. Our comprehensive regulatory maps offer insights into the mechanisms by which folate may prevent NTDs. Lastly, our distal regulatory maps provide a better understanding of the potential role of neurological-disorder-associated SNPs.</p

Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation

Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation.</p

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3(K392R,) on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3(K392R) and healthy-control cells, but in later stages, NEUROG3 expressionwas upregulated prematurely in STAT3(K392R) cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3(K392R) -activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3(K392R). Collectively, our results demonstrate that the STAT3(K392R) mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.Peer reviewe

Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential

Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other. However, genetic variation imparts a donor-specific expression and methylation profile in reprogrammed cells that leads to variable functional capacities of iPSC lines. Our results suggest that integration-free, bona fide iPSC lines from fibroblasts and blood can be combined in repositories to form biobanks. Due to the impact of genetic variation on iPSC differentiation, biobanks should contain cells from large numbers of donors.Peer reviewe

Gene expression profile of the hippocampus of a behavioural model of depression

Although the neurobiological basis of depression has not been fully elucidated, numerous studies have emphasized that in the etiology of depression stress may be the most significant cause, together with genetic vulnerability. Stress induces a coordinated and complex response that is adaptive and integral to survival. The brain's ability to adapt and change over time is refered to neuroplasticity and long-term plasticity in the brain requires changes in gene expression. However, exposure to intense or chronic stressors leads to an increased risk for the development of stress-related disorders including major depression. Numerous studies demonstrate that neuronal atrophy and loss of plasticity occur in hippocampus in response to stress and depression. Therefore, the hippocampal region may play a central role in depressive illness. Likewise changes in gene expression underlying the plasticity of hippocampal structures appear to be relevant in undenstanding the molecular and cellular mechanisms involved in the etiology as well as the treatment of depression, and the mechanisms leading vulnerability or resilience to stress. In fact, humans display a remarkable heterogeneity in their responses to stress and adversity. Although we are beginning to understand how maladaptive neurobiological changes may contribute to depression, relatively little is known about the molecular mechanisms that may underlie stress resilience. Here we set out to investigate the changes in the gene expression profile underlying the effects of stress on the hippocampus using a behavioural paradigm of depression, the chronic escape deficit model [1], which is based on the modified reactivity of rats to external stimuli, the escape deficit, induced by exposure to intense and unavoidable stress. The chronic escape deficit model starts as an acute escape deficit which can be indefinitely sustained by repeated administration of mild stressors. This approach has proved to be a valid and useful model of depression because it consider depressive symptoms like behavioural despair. We performed gene expression profiling in the rat hippocampus, using GeneChip Rat Exon Array (Affymetrix). Using this new platform we carried out analyses of gene expression on three different levels: gene, transcript and exon level analyses. The behavioural results showed that exposure to intense and unavoidable stressful procedure induced escape deficit only in 60% of them. Whereas the animals remaining display a behaviour apparently identical to control animals which did not undergo the stressful procedure. Comparing gene expression profiles and performing functional analysis on differently expressed genes we have indicated multiple pathways that may be involved in the underlying mechanisms of stress condition associated with escape deficit. Moreover we identified possible cellular functions and biological processes that could represent targets that may contribute to mediate the effects of stress on the hippocampal plasticity. Such as, gene expression profiling of stress-vulnerable and stress-resilient animals revealed distinct transcriptional profiles, suggesting that resilient behaviour represents an active neurobiological process and not simply the absence of vulnerability

Microarray analysis in hippocampus of rats treated with escitalopram in the chronic escape deficit model of depression

Currently, the biological bases of depression and the molecular mechanisms underlying antidepressant action are not completely understood. Valuable tools to better understand the pathophysiology of this disease are behavioural models of depression eventually combined with genome-wide gene expression analysis. The Chronic Escape Deficit (CED) is a validated behavioural model of depression, based on the induction of an escape deficit after exposure of rats to an unavoidable stress. This model allows to evaluate the capacity of a treatment to revert the escape deficit. The antidepressant drugs tested in CED model need to be administered for at least 3−4 weeks in order to revert the escape deficit [1,2]. In this study, we demonstrated that already after one week of treatment with Escitalopram, a widely used SSRI, 50% of the animals responded reverting the escape deficit induced. Moreover, the other 50% of treated animals did not respond also after 3−4 weeks of treatment. Since in the CED model the behavioural alteration is induced by stress application and reverted by escitalopram treatment in only half of animals, the aims of our study were two fold: (i) to investigate transcriptional changes activated by stress; (ii) to study the different gene expression pattern involved into mechanisms of the response and not response to the pharmacological treatment. To address these issues we performed a microarray experiment in the rat hippocampus using Affymetrix GeneChip Rat Exon 1.0 ST evaluating both gene-level and exon-level expression profiling on the whole genome. Total RNA extracted from hippocampus of each animal was utilized to chip a single array using the Affymetrix protocols. 20 single arrays were utilized for data analysis and divided into five replicates for each experimental group (control, stress, stress-escitalopram responders and stress escitalopram-not responders). Using two parallel analyses (gene level and exon level) of raw data files carried out in Expression Console software using iterPLIER algorithms, we identified genes and exons that were differentially regulated in each pairwise comparison considered. The exons identified in this study were examinated for their biological association to gene ontology (GO) categories using eGOn software. Moreover, all exons differentially expressed were also uploaded into Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) in order to identify molecular pathways and functions related to stress and escitalopram response. Our results suggest that stress may exert a negative effect on gene transcription since the largest number of genes was downregulated. Moreover from our data it seems that a different pattern of gene expression exhibits between animals that respond and that did not respond to escitalopram treatment. Functional analysis of exon dataset, arising from stress protocol and escitalopram treatment, reflects interesting different biological features. More specifically, the biological functions regard both molecular and cellular functions, such as cellular growth and proliferation, gene expression and signal transduction, as well as involvement of central neurotransmission and immune response. We believe that this pharmacogenomic approach will be helpful to understand the molecular mechanisms involved in the pathogenesis of depression as well as in the response to antidepressant drugs

Additional file 1: of cChIP-seq: a robust small-scale method for investigation of histone modifications

The following additional data are available with the online version of this paper. Additional data file 1 contains the following figures. Figure S1. illustrates the optimization on sonication down to 30,000 crosslinked cells using the Covaris LE220 ultrasonicator. Figure S2. provides p-values for the Pearson’s correlation heatmaps shown in Figs. 2, 3 and 4. Figure S3. relates to Fig. 2 and provides additional results for H3K4me3 in K562 cell line. Figure S4. relates to Fig. 3 and provides additional results for H3K4me1 in K562 cell line. Figure S5. shows results obtained for H3K4me1 in H1 hESC line. (PDF 3493 kb

Transcriptional profiles underlying vulnerability and resilience in rats exposed to an acute unavoidable stress

A complex interplay between gene and environment influences the vulnerability or the resilience to stressful events. In the acute escape deficit (AED) paradigm, rats exposed to an acute unavoidable stress (AUS) develop impaired reactivity to noxious stimuli. Here we assessed the behavioral and molecular changes in rats exposed to AUS. A genome-wide microarray experiment generated a comprehensive picture of changes in gene expression in the hippocampus and the frontal cortex of animals exposed or not to AUS. Exposure to AUS resulted in two distinct groups of rats with opposite behavioral profiles: one developing an AED, called "stress vulnerable," and one that did not develop an AED, called "stress resilient." Genome-wide profiling revealed a low percentage of overlapping mechanisms in the two areas, suggesting that, in the presence of stress, resilience or vulnerability to AUS is sustained by specific changes in gene expression that can either buffer or promote the behavioral and molecular adverse consequences of stress. Specifically, we observed in the frontal cortex a downregulation of the transcript coding for interferon-β and leukemia inhibitory factor in resilient rats and an upregulation of neuroendocrine related genes, growth hormone and prolactin, in vulnerable rats. In the hippocampus, the muscarinic M2 receptor was downregulated in vulnerable but upregulated in resilient rats. Our findings demonstrate that opposite behavioral responses did not correspond to opposite regulatory changes of the same genes, but resilience rather than vulnerability to stress was associated with specific changes, with little overlap, in the expression of patterns of genes. © 2012 Wiley Periodicals, Inc.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe