Potential Key Regulators Research Articles

Reprogramming cell fate with a genome-scale library of artificial transcription factors

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Mergeomics: multidimensional data integration to identify pathogenic perturbations to biological systems.

BackgroundComplex diseases are characterized by multiple subtle perturbations to biological processes. New omics platforms can detect these perturbations, but translating the diverse molecular and statistical information into testable mechanistic hypotheses is challenging. Therefore, we set out to create a public tool that integrates these data across multiple datasets, platforms, study designs and species in order to detect the most promising targets for further mechanistic studies.ResultsWe developed Mergeomics, a computational pipeline consisting of independent modules that 1) leverage multi-omics association data to identify biological processes that are perturbed in disease, and 2) overlay the disease-associated processes onto molecular interaction networks to pinpoint hubs as potential key regulators. Unlike existing tools that are mostly dedicated to specific data type or settings, the Mergeomics pipeline accepts and integrates datasets across platforms, data types and species. We optimized and evaluated the performance of Mergeomics using simulation and multiple independent datasets, and benchmarked the results against alternative methods. We also demonstrate the versatility of Mergeomics in two case studies that include genome-wide, epigenome-wide and transcriptome-wide datasets from human and mouse studies of total cholesterol and fasting glucose. In both cases, the Mergeomics pipeline provided statistical and contextual evidence to prioritize further investigations in the wet lab. The software implementation of Mergeomics is freely available as a Bioconductor R package.ConclusionMergeomics is a flexible and robust computational pipeline for multidimensional data integration. It outperforms existing tools, and is easily applicable to datasets from different studies, species and omics data types for the study of complex traits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3198-9) contains supplementary material, which is available to authorized users.

MicroRNAs role as potential biomarkers and key regulators in melanoma.

Malignant melanoma (MM) is a highly aggressive skin cancer with high incidence worldwide. It originates from melanocytes and is characterized by invasion, early metastasis and despite the use of new drugs it is still characterized by high mortality. Since an early diagnosis determines a better prognosis, it is important to explore novel prognostic markers in the management of patients with MM. microRNAs (miRNAs) are small (∼22 nucleotides) single-stranded non-coding RNAs that negatively regulate the expression of more than 60% of human genes.miRNAs alterations are involved in several cancers, including MM, where a differential expression for some of them has been reported between healthy controls and MM patients. Moreover, since miRNAs are stable and easily detectable in body fluids, they might be considered as robust candidate biomarkers useful to identify risk of MM, to diagnose an early lesion and/or an early metastatic disease. This review highlights the importance of miRNAs as risk factors, prognostic factors and their role as molecular regulator in the development and progression of MM. © 2016 Wiley Periodicals, Inc.

The Arabidopsis phytohormone crosstalk network involves a consecutive metabolic route and circular control units of transcription factors that regulate enzyme-encoding genes.

BackgroundPhytohormone synergies and signaling interdependency are important topics in plant developmental biology. Physiological and genetic experimental evidence for phytohormone crosstalk has been accumulating and a genome-scale enzyme correlation model representing the Arabidopsis metabolic pathway has been published. However, an integrated molecular characterization of phytohormone crosstalk is still not available.ResultsA novel modeling methodology and advanced computational approaches were used to construct an enzyme-based Arabidopsis phytohormone crosstalk network (EAPCN) at the biosynthesis level. The EAPCN provided the structural connectivity architecture of phytohormone biosynthesis pathways and revealed a surprising result; that enzymes localized at the highly connected nodes formed a consecutive metabolic route. Furthermore, our analysis revealed that the transcription factors (TFs) that regulate enzyme-encoding genes in the consecutive metabolic route formed structures, which we describe as circular control units operating at the transcriptional level. Furthermore, the downstream TFs in phytohormone signal transduction pathways were found to be involved in the circular control units that included the TFs regulating enzyme-encoding genes. In addition, multiple functional enzymes in the EAPCN were found to be involved in ion and pH homeostasis, environmental signal perception, cellular redox homeostasis, and circadian clocks. Last, publicly available transcriptional profiles and a protein expression map of the Arabidopsis root apical meristem were used as a case study to validate the proposed framework.ConclusionsOur results revealed multiple scales of coupled mechanisms in that hormonal crosstalk networks that play a central role in coordinating internal developmental processes with environmental signals, and give a broader view of Arabidopsis phytohormone crosstalk. We also uncovered potential key regulators that can be further analyzed in future studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0333-9) contains supplementary material, which is available to authorized users.

Transcriptome landscape of perennial wild Cicer microphyllum uncovers functionally relevant molecular tags regulating agronomic traits in chickpea.

The RNA-sequencing followed by de-novo transcriptome assembly identified 11621 genes differentially xpressed in roots vs. shoots of a wild perennial Cicer microphyllum. Comparative analysis of transcriptomes between microphyllum and cultivated desi cv. ICC4958 detected 12772 including 3242 root- and 1639 shoot-specific microphyllum genes with 85% expression validation success rate. Transcriptional reprogramming of microphyllum root-specific genes implicates their possible role in regulating differential natural adaptive characteristics between wild and cultivated chickpea. The transcript-derived 5698 including 282 in-silico polymorphic SSR and 127038 SNP markers annotated at a genome-wide scale exhibited high amplification and polymorphic potential among cultivated (desi and kabuli) and wild accessions suggesting their utility in chickpea genomics-assisted breeding applications. The functional significance of markers was assessed based on their localization in non-synonymous coding and regulatory regions of microphyllum root-specific genes differentially expressed predominantly in ICC 4958 roots under drought stress. A high-density 490 genic SSR- and SNP markers-anchored genetic linkage map identified six major QTLs regulating drought tolerance-related traits, yield per plant and harvest-index in chickpea. The integration of high-resolution QTL mapping with comparative transcriptome profiling delineated five microphyllum root-specific genes with non-synonymous and regulatory SNPs governing drought-responsive yield traits. Multiple potential key regulators and functionally relevant molecular tags delineated can drive translational research and drought tolerance-mediated chickpea genetic enhancement.

Differential transcriptomic responses to Fusarium graminearum infection in two barley quantitative trait loci associated with Fusarium head blight resistance.

BackgroundFusarium graminearum causes Fusarium head blight (FHB), a major disease problem worldwide. Resistance to FHB is controlled by quantitative trait loci (QTL) of which two are located on barley chromosomes 2H bin8 and 6H bin7. The mechanisms of resistance mediated by FHB QTL are poorly defined.ResultsNear-isogenic lines (NILs) carrying Chevron-derived resistant alleles for the two QTL were developed and exhibited FHB resistance in field trials. To understand the molecular responses associated with resistance, transcriptomes of the NILs and recurrent parents (M69 and Lacey) were investigated with RNA sequencing (RNA-Seq) after F. graminearum or mock inoculation. A total of 2083 FHB-responsive transcripts were detected and provide a gene expression atlas for the barley-F. graminearum interaction. Comparative analysis of the 2Hb8 resistant (R) NIL and M69 revealed that the 2Hb8 R NIL exhibited an elevated defense response in the absence of fungal infection and responded quicker than M69 upon fungal infection. The 6Hb7 R NIL displayed a more rapid induction of a set of defense genes than Lacey during the early stage of fungal infection. Overlap of differentially accumulated genes were identified between the two R NILs, suggesting that certain responses may represent basal resistance to F. graminearum and/or general biotic stress response and were expressed by both resistant genotypes. Long noncoding RNAs (lncRNAs) have emerged as potential key regulators of transcription. A total of 12,366 lncRNAs were identified, of which 604 were FHB responsive.ConclusionsThe current transcriptomic analysis revealed differential responses conferred by two QTL during F. graminearum infection and identified genes and lncRNAs that were associated with FHB resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2716-0) contains supplementary material, which is available to authorized users.

Reconstruction of gene regulatory networks reveals chromatin remodelers and key transcription factors in tumorigenesis.

BackgroundAlterations in genetic and epigenetic landscapes are known to contribute to the development of different types of cancer. However, the mechanistic links between transcription factors and the epigenome which coordinate the deregulation of gene networks during cell transformation are largely unknown.MethodsWe used an isogenic model of stepwise tumorigenic transformation of human primary cells to monitor the progressive deregulation of gene networks upon immortalization and oncogene-induced transformation. We applied a systems biology approach by combining transcriptome and epigenome data for each step during transformation and integrated transcription factor–target gene associations in order to reconstruct the gene regulatory networks that are at the basis of the transformation process.ResultsWe identified 142 transcription factors and 24 chromatin remodelers/modifiers (CRMs) which are preferentially associated with specific co-expression pathways that originate from deregulated gene programming during tumorigenesis. These transcription factors are involved in the regulation of divers processes, including cell differentiation, the immune response, and the establishment/modification of the epigenome. Unexpectedly, the analysis of chromatin state dynamics revealed patterns that distinguish groups of genes which are not only co-regulated but also functionally related. Decortication of transcription factor targets enabled us to define potential key regulators of cell transformation which are engaged in RNA metabolism and chromatin remodeling.ConclusionsWe reconstructed gene regulatory networks that reveal the alterations occurring during human cellular tumorigenesis. Using these networks we predicted and validated several transcription factors as key players for the establishment of tumorigenic traits of transformed cells. Our study suggests a direct implication of CRMs in oncogene-induced tumorigenesis and identifies new CRMs involved in this process. This is the first comprehensive view of the gene regulatory network that is altered during the process of stepwise human cellular tumorigenesis in a virtually isogenic system.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0310-3) contains supplementary material, which is available to authorized users.

Weighted enrichment method for prediction of transcription regulators from transcriptome and global chromatin immunoprecipitation data.

Predicting responsible transcription regulators on the basis of transcriptome data is one of the most promising computational approaches to understanding cellular processes and characteristics. Here, we present a novel method employing vast amounts of chromatin immunoprecipitation (ChIP) experimental data to address this issue. Global high-throughput ChIP data was collected to construct a comprehensive database, containing 8 578 738 binding interactions of 454 transcription regulators. To incorporate information about heterogeneous frequencies of transcription factor (TF)-binding events, we developed a flexible framework for gene set analysis employing the weighted t-test procedure, namely weighted parametric gene set analysis (wPGSA). Using transcriptome data as an input, wPGSA predicts the activities of transcription regulators responsible for observed gene expression. Validation of wPGSA with published transcriptome data, including that from over-expressed TFs, showed that the method can predict activities of various TFs, regardless of cell type and conditions, with results totally consistent with biological observations. We also applied wPGSA to other published transcriptome data and identified potential key regulators of cell reprogramming and influenza virus pathogenesis, generating compelling hypotheses regarding underlying regulatory mechanisms. This flexible framework will contribute to uncovering the dynamic and robust architectures of biological regulation, by incorporating high-throughput experimental data in the form of weights.

Genome-wide DNA methylation profile of developing deciduous tooth germ in miniature pigs.

BackgroundDNA methylation is an important epigenetic modification critical to the regulation of gene expression during development. To date, little is known about the role of DNA methylation in tooth development in large animal models. Thus, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in E50 and E60 tooth germ from miniature pigs using methylated DNA immunoprecipitation-sequencing (MeDIP-seq).ResultsWe observed different DNA methylation patterns during the different developmental stages of pig tooth germ. A total of 2469 differentially methylated genes were identified. Functional analysis identified several signaling pathways and 104 genes that may be potential key regulators of pig tooth development from E50 to E60.ConclusionsThe present study provided a comprehensive analysis of the global DNA methylation pattern of tooth germ in miniature pigs and identified candidate genes that potentially regulate tooth development from E50 to E60.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2485-9) contains supplementary material, which is available to authorized users.

Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats.

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets.

Comprehensive Expression of Wnt Signaling Pathway Genes during Development and Maturation of the Mouse Cochlea.

BackgroundIn the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points.ResultsWe identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity.ConclusionA large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.

Interactome analysis of gene expression profile reveals potential novel key transcriptional regulators of skin pathology in vitiligo.

Selective destruction of epidermal melanocytes is central to vitiligo (VL), a common acquired, autoimmune depigmentory disorder of the skin. Like other autoimmune diseases, the pathogenesis of VL is obscure and both multifactorial and polygenic. The prevailing theory is that VL may be part of an autoimmune diathesis. To evaluate mechanisms underlying disease development and progression, we studied genome-wide gene expression from lesional and non-lesional skin of patients with non-segmental VL. Unbiased clustering and principal components analyses reveals a 'lesional pathology'-based signature. Pathway-based analyses of the differentially expressed genes underscore processes such as melanocyte development and cell cycle as central drivers of the disease state. Interactome analysis identifies several key transcriptional regulators potentially affecting disease pathogenesis both within and 'hidden' from the data set. Finally, two genes within six identified transcriptional 'hot spots' coincide with previous VL-associated genetic elements. The remaining genes in the 'hot spots' offer an additional set of potential disease-linked loci that may help to guide future studies aimed at identifying disease risk genes.

Abstract 164: Long noncoding RNAs in the breast of healthy women are detected in different subtypes of breast cancer

Abstract Long non-coding RNA (lncRNAs) are RNA molecules longer than 200 nucleotides that are not translated into proteins, but regulate the transcription of genes involved in different cellular processes, including differentiation, cancer initiation and progression. The relevance of lncRNAs in the transcription field is beginning to be explored and their roles have been found to vary from guiding proteins to the genome to scaffolding proteins complexes needed for the transcription of a specific gene. Microarray analysis of normal breast of parous and nulliparous postmenopausal women revealed that lncRNAs are up-regulated in the parous breast [Int. J. Cancer: 131, 1059-1070, 2012]. Subsequently, RNA sequencing was performed to a subset of the samples, originally analyzed by microarray, to understand the role of lncRNAs in the regulation of transcription and their potential function in pregnancy's preventive effect in reducing the lifetime risk of developing breast cancer. In this work, RNA sequencing of healthy postmenopausal breast tissue biopsies from 8 parous and 8 nulliparous women using Illumina platform was performed. The sequencing results show that there are 42 lncRNAs differentially expressed between parous and nulliparous breast tissue. After analysis of these 42 lncRNAs using bioinformatics and thermodynamic filters, 11 lncRNAs were selected to be tested in vitro. Using RT-qPCR we have determined that the expression of these 11 lncRNAs is characteristic of each epithelial breast cancer cell subtype (luminal, basal and cells that overexpress HER2). These results indicate that these lncRNAs may be key targets to specifically study and potentially treat different types of breast cancers. Moreover, these lncRNAs are differentially expressed when breast tumor tissue and its adjacent normal tissue are compared. Consequently, the results in cancer/normal-like cell lines and tumor/adjacent normal tissue point to the potential of these lncRNAs as targets, not only for therapeutic approaches against breast cancer, but also as preventive measures for nulliparous women. This work provides novel information on lncRNAs differentially expressed in breast cells when comparing pregnancy versus the lack of a full term pregnancy, and also places lncRNAs as potential key regulators in cancer initiation and cancer progression. Determining their functions in both differentiation and cancer will render a comprehensive picture of their role in the human breast at the transcriptomic level. Furthermore, the manipulation of these lncRNAs can eventually lead to the development of therapeutics for breast cancer prevention. (This work was supported by NIH core grant CA06927 to Fox Chase Cancer Center) Citation Format: Maria Barton, Julia Santucci-Pereira, Theresa D. Nguyen, Jose Russo. Long noncoding RNAs in the breast of healthy women are detected in different subtypes of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 164. doi:10.1158/1538-7445.AM2015-164

Network analysis of temporal functionalities of the gut induced by perturbations in new-born piglets.

BackgroundEvidence is accumulating that perturbation of early life microbial colonization of the gut induces long-lasting adverse health effects in individuals. Understanding the mechanisms behind these effects will facilitate modulation of intestinal health. The objective of this study was to identify biological processes involved in these long lasting effects and the (molecular) factors that regulate them. We used an antibiotic and the same antibiotic in combination with stress on piglets as an early life perturbation. Then we used host gene expression data from the gut (jejunum) tissue and community-scale analysis of gut microbiota from the same location of the gut, at three different time-points to gauge the reaction to the perturbation. We analysed the data by a new combination of existing tools. First, we analysed the data in two dimensions, treatment and time, with quadratic regression analysis. Then we applied network-based data integration approaches to find correlations between host gene expression and the resident microbial species.ResultsThe use of a new combination of data analysis tools allowed us to identify significant long-lasting differences in jejunal gene expression patterns resulting from the early life perturbations. In addition, we were able to identify potential key gene regulators (hubs) for these long-lasting effects. Furthermore, data integration also showed that there are a handful of bacterial groups that were associated with temporal changes in gene expression.ConclusionThe applied systems-biology approach allowed us to take the first steps in unravelling biological processes involved in long lasting effects in the gut due to early life perturbations. The observed data are consistent with the hypothesis that these long lasting effects are due to differences in the programming of the gut immune system as induced by the temporary early life changes in the composition and/or diversity of microbiota in the gut.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1733-8) contains supplementary material, which is available to authorized users.

Epigenetic and molecular signatures of cord blood CD34+ cells treated with histone deacetylase inhibitors

Epigenetic modifications tightly regulate the gene expression and cellular function of haematopoietic stem cells. Histone deacetylase inhibitors (HDACIs) alter the gene expression profile of cord blood (CB) CD34(+) cells by controlling the genes involved in chromatin modification, thereby influencing the self-renewal, maintenance and expansion of haematopoietic stem and progenitor cells (HSPCs). The class I and II HDACIs, valproic acid and scriptaid, were utilized to expand CB-CD34(+) cells ex vivo. The gene profiling was performed on HSPC using Illumina microarray, GeneGO MetaCore(™) and Ingenuity pathway analyses. The molecular analyses were performed using Q-PCR and Western blotting. Each HDACI treatment of CB-CD34(+) cells created unique epigenetic and molecular signatures that governed chromatin modification required for cellular and functional behaviour of stem cells. GeneGO MetaCore(™) and Ingenuity pathway analyses established the molecular understanding of epigenetically regulated HSPCs in the presence of scriptaid and VPA that revealed different network(s) of potential regulators during erythropoiesis. VPA induced transcriptional activation of the glucocorticoid receptor (GCR) and an increase in the intracellular signalling of signal transducers and activators of transcription (STAT) required during stress erythropoiesis. Canonical Wnt signalling and many epigenetically regulated chromatin remodellers were significantly influenced so as to establish maintenance and regulation of HSPC. Treatment with Individual HDACIs has demonstrated significantly unique epigenetic and molecular signatures of CB-HSPC. This study identifies potential key regulators of HSPC and gives insights into the clinically important processes of HSPC expansion and haematopoietic lineage development for transplantation purposes.

Combined QTL and selective sweep mappings with coding SNP annotation and cis-eQTL analysis revealed PARK2 and JAG2 as new candidate genes for adiposity regulation.

Very few causal genes have been identified by quantitative trait loci (QTL) mapping because of the large size of QTL, and most of them were identified thanks to functional links already known with the targeted phenotype. Here, we propose to combine selection signature detection, coding SNP annotation, and cis-expression QTL analyses to identify potential causal genes underlying QTL identified in divergent line designs. As a model, we chose experimental chicken lines divergently selected for only one trait, the abdominal fat weight, in which several QTL were previously mapped. Using new haplotype-based statistics exploiting the very high SNP density generated through whole-genome resequencing, we found 129 significant selective sweeps. Most of the QTL colocalized with at least one sweep, which markedly narrowed candidate region size. Some of those sweeps contained only one gene, therefore making them strong positional causal candidates with no presupposed function. We then focused on two of these QTL/sweeps. The absence of nonsynonymous SNPs in their coding regions strongly suggests the existence of causal mutations acting in cis on their expression, confirmed by cis-eQTL identification using either allele-specific expression or genetic mapping analyses. Additional expression analyses of those two genes in the chicken and mice contrasted for adiposity reinforces their link with this phenotype. This study shows for the first time the interest of combining selective sweeps mapping, coding SNP annotation and cis-eQTL analyses for identifying causative genes for a complex trait, in the context of divergent lines selected for this specific trait. Moreover, it highlights two genes, JAG2 and PARK2, as new potential negative and positive key regulators of adiposity in chicken and mice.

Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study. RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment. In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

Spinal cord injury and the neuron-intrinsic regeneration-associated gene program.

Spinal cord injury (SCI) affects millions of people worldwide and causes a significant physical, emotional, social and economic burden. The main clinical hallmark of SCI is the permanent loss of motor, sensory and autonomic function below the level of injury. In general, neurons of the central nervous system (CNS) are incapable of regeneration, whereas injury to the peripheral nervous system is followed by axonal regeneration and usually results in some degree of functional recovery. The weak neuron-intrinsic regeneration-associated gene (RAG) response upon injury is an important reason for the failure of neurons in the CNS to regenerate an axon. This response consists of the expression of many RAGs, including regeneration-associated transcription factors (TFs). Regeneration-associated TFs are potential key regulators of the RAG program. The function of some regeneration-associated TFs has been studied in transgenic and knock-out mice and by adeno-associated viral vector-mediated overexpression in injured neurons. Here, we review these studies and propose that AAV-mediated gene delivery of combinations of regeneration-associated TFs is a potential strategy to activate the RAG program in injured CNS neurons and achieve long-distance axon regeneration.

Abstract 1480: Long noncoding RNAs in the postmenopausal breast and their role in cancer prevention

Abstract Our initial transcriptomic analysis in the normal breast of parous and nulliparous women reveals that long non-coding RNA genes (lncRNAs) such as nuclear paraspeckle assembly transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and X inactive specific transcript (XIST) are up-regulated in the parous breast [Cancer Prev Res 4 1457-1464, 2011, BMC Medical Genomics. 5:46. 2012 and Int. J. Cancer: 131, 1059-1070, 2012]. This novel observation provides a new paradigm in our understanding of the role of lncRNAs in the regulation of transcription and its potential function in pregnancy's preventive effect in reducing the lifetime risk of developing breast cancer. In the present work, we performed RNA sequencing of healthy postmenopausal breast tissue biopsies from 8 parous (P) and 8 nulliparous (NP) women using Illumina TruSeq for the library preparation and Illumina HiSeq 2000 (Illumina Inc., San Diego, CA) for RNA sequencing (RNAseq). The 16 sequenced samples (8P, 8NP) satisfied quality control thresholds. The sequencing results show that 140 genes were differentially expressed in the parous vs. the nulliparous breast tissue with fold change of two and a p-value of 0.05. Also, using t-statistics with p-value less than 0.01 and fold-change of at least two as criteria of significance, we identified 42 differentially expressed lncRNAs between P and NP women. Of these, 21 lncRNAs were up-regulated and 21 were down-regulated. We also identified 10 lncRNAs that show strong Pearson correlation of the differentially expressed lncRNA and its corresponding gene, for example lnc-CDH5-1 (long non-coding CDH5, transcript variant 1) and its corresponding gene CDH5 (cadherin 5, type 2) show a correlation p- value of 0.017 and a correlation estimate of -0.59. These results provide not only novel information on the hormonal regulation of lncRNAs induced by pregnancy in breast cells, but also place lncRNAs as potential key regulators in breast differentiation. These results begin to render a comprehensive picture of the differentiation of the human breast at the transcriptomic level, findings that will lead to the identification of the role played by lncRNAs in the prevention of breast cancer. (The sample collection was supported by Avon Foundation for Women Breast Cancer Research Program grant 02-2010-117 and the RNA sequencing studies by NIH core grant CA06927 to Fox Chase Cancer Center). Citation Format: Maria Barton, Julia Santucci-Pereira, Ricardo Lopez de Cicco, Irma H. Russo, Eric A. Ross, Michael Slifker, Suraj Peri, Pal Bordas, Per Lenner, Göran Hallmans, Paolo Toniolo, Jose Russo. Long noncoding RNAs in the postmenopausal breast and their role in cancer prevention. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2014-1480

Genome-Scale Transcriptomic Insights into Molecular Aspects of Abiotic Stress Responses in Chickpea

Chickpea is an important legume crop plant and various abiotic stresses are the major constraints affecting its overall productivity. For discovery of candidate genes involved in abiotic stress responses, we employed RNA sequencing for transcriptome profiling of roots and shoots of chickpea seedlings subjected to desiccation, salinity, and cold stresses. In total, we generated more than 250 million high-quality reads from non-stressed and stressed tissue samples. Data analyses provided a comprehensive view of the dynamic transcriptional response of chickpea tissues to different abiotic stresses. Differential expression analysis identified a total of 11,640 chickpea transcripts showing response to at least one of the stress conditions. The reference-based transcriptome assembly was generated and at least 3,536 previously unannotated gene loci differentially expressed under abiotic stress conditions were identified. We observed extensive transcriptional reprogramming of genes involved in transcription regulation, energy metabolism, photosynthesis, hormonal responses, secondary metabolite biosynthesis and osmoprotectant metabolism under stress conditions. In addition, genes involved in post-translational modifications, RNA metabolic processes, and epigenetic regulation were also significantly highlighted. The comprehensive transcriptome analyses presented in this study revealed several potential key regulators of plant response to abiotic stresses and open avenues to carry out functional and applied genomic studies for improving abiotic stress tolerance in chickpea.