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1.  Alcohol induced epigenetic alterations to developmentally crucial genes regulating neural stemness and differentiation 
From studies using a diverse range of model organisms, we now acknowledge that epigenetic changes to chromatin structure provide a plausible link between environmental teratogens and alterations in gene expression leading to disease. Observations from a number of independent laboratories indicate ethanol has the capacity to act as a powerful epigenetic disruptor and potentially derail the coordinated processes of cellular differentiation. In this study, we sought to examine whether primary neurospheres cultured under conditions maintaining stemness were susceptible to alcohol-induced alterations of the histone code. We focused our studies on trimethylated histone 3 lysine 4 and trimethylated histone 3 lysine 27, as these are two of the most prominent post-translational histone modifications regulating stem cell maintenance and neural differentiation.
Primary neurosphere cultures were maintained under conditions promoting the stem cell state and treated with ethanol for five days. Control and ethanol treated cellular extracts were examined using a combination of quantitative RT-PCR and chromatin immunoprecipitation techniques.
We find that the regulatory regions of genes controlling both neural precursor cell identity and processes of differentiation exhibited significant declines in the enrichment of the chromatin marks examined. Despite these widespread changes in chromatin structure, only a small subset of genes including Dlx2, Fabp7, Nestin, Olig2, and Pax6 displayed ethanol induced alterations in transcription. Unexpectedly, the majority of chromatin modifying enzymes examined including members of the Polycomb Repressive Complex displayed minimal changes in expression and localization. Only transcripts encoding Dnmt1, Uhrf1, Ehmt1, Ash2l, Wdr5, and Kdm1b exhibited significant differences.
Our results indicate primary neurospheres maintained as stem cells in vitro are susceptible to alcohol-induced perturbation of the histone code and errors in the epigenetic program. These observations indicate that alterations to chromatin structure may represent a crucial component of alcohol teratogenesis and progress towards a better understanding of the developmental origins of FASDs.
PMCID: PMC3688681  PMID: 23488822
Epigenetic Programming; Bivalent Genes; Chromatin; Fetal Alcohol Syndrome; Neural Stem Cells
2.  Generation of Trophoblast Stem Cells 
The isolation and culture of both embryonic and extraembryonic stem cells provide an enormous opportunity to study the molecular processes that establish and maintain lineage-specific, monoallelic patterns of gene expression. This chapter describes the isolation an culture of trophectoderm stem cells from mouse blastocyst stage embryos. Using this powerful in vitro system, scientists can now begin to tease apart the epigenetic processes that result in placental patterns of imprinted gene expression and begin to better understand the role these genes play in development and disease.
PMCID: PMC4061734  PMID: 22907489
Placental stem cell; Trophectoderm; Extraembryonic lineage; TS cell
3.  Reshaping the Transcriptional Frontier: Epigenetics and Somatic Cell Nuclear Transfer 
Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.
PMCID: PMC3953569  PMID: 24167064
4.  Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure 
Alcohol (Fayetteville, N.Y.)  2013;47(2):109-120.
Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean CT values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens.
PMCID: PMC3653297  PMID: 23317542
Reference gene; Normalization; Alcohol; Quantitative RT-PCR; Real-time PCR; Stem cells
5.  Prenatal Alcohol Exposure and Cellular Differentiation 
Exposure to alcohol significantly alters the developmental trajectory of progenitor cells and fundamentally compromises tissue formation (i.e., histogenesis). Emerging research suggests that ethanol can impair mammalian development by interfering with the execution of molecular programs governing differentiation. For example, ethanol exposure disrupts cellular migration, changes cell–cell interactions, and alters growth factor signaling pathways. Additionally, ethanol can alter epigenetic mechanisms controlling gene expression. Normally, lineage-specific regulatory factors (i.e., transcription factors) establish the transcriptional networks of each new cell type; the cell’s identity then is maintained through epigenetic alterations in the way in which the DNA encoding each gene becomes packaged within the chromatin. Ethanol exposure can induce epigenetic changes that do not induce genetic mutations but nonetheless alter the course of fetal development and result in a large array of patterning defects. Two crucial enzyme complexes—the Polycomb and Trithorax proteins—are central to the epigenetic programs controlling the intricate balance between self-renewal and the execution of cellular differentiation, with diametrically opposed functions. Prenatal ethanol exposure may disrupt the functions of these two enzyme complexes, altering a crucial aspect of mammalian differentiation. Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder.
PMCID: PMC3860417  PMID: 24313167
Alcohol exposure; ethanol exposure; prenatal alcohol exposure; prenatal alcohol exposure; fetal alcohol spectrum disorders; fetal alcohol syndrome (FAS); FAS phenotypes; fetal development; epigenetics; epigenetic mechanisms; epigenetic changes; gene expression; developmental programming; transcription; cellular differentiation; Polycomb group proteins; Trithorax group proteins
6.  Examination of DNA Methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development 
In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for clone embryonic development to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes and genomic DNA hypermethylation are frequently observed in reconstructed embryos suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine whether the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogentic activation and SCNT. By the 8-Cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos; likely in response to the state of genomic hypermethylation, while the de novo methyltranserases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic / maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development and suggest proper transcriptional reprogramming of this gene family in SCNT embryos.
PMCID: PMC3095725  PMID: 21480430
Epigenetics; DNA Methylation; Somatic Cell Nuclear Transfer; DNA Methyltransferase; Preimplantation Development
7.  Selection of Stable Reference Genes for Quantitative RT-PCR Comparisons of Mouse Embryonic and Extra-Embryonic Stem Cells 
PLoS ONE  2011;6(11):e27592.
Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.
Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.
Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.
PMCID: PMC3213153  PMID: 22102912
8.  Profiling Essential Genes in Human Mammary Cells by Multiplex RNAi Screening 
Science (New York, N.Y.)  2008;319(5863):617-620.
By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line–specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities.
PMCID: PMC2981861  PMID: 18239125
9.  The PcG Gene Sfmbt2 is Paternally Expressed in Extraembryonic Tissues 
Gene expression patterns : GEP  2007;8(2):107-116.
Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC.
PMCID: PMC2220043  PMID: 18024232
Genomic Imprinting; Sfmbt2; PcG gene; Extraembryonic tissues; placenta; MMU2; YY1; microarrays; uniparental embryos

Results 1-9 (9)