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Systematic analysis of gene regulatory networks

基因调控网络的系统分析

基本信息

批准号：
9341859
负责人：
David Schlessinger
金额：
$ 16.51万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9341859
关键词：
Aging Algorithms Alkaline Phosphatase Analysis of Variance Behavior Binding Sites Biological Assay Cell Differentiation process Cell Lineage Cells Chimera organism Computer software DNA Microarray Chip Data Data Analyses Databases Doxycycline ES Cell Line Embryo Equilibrium Expressed Sequence Tags Flow Cytometry Gene Expression Gene Expression Profile Gene Targeting Genes Germ Layers Glass Goals Hour Immunohistochemistry In Situ In Situ Hybridization In Vitro Individual Injection of therapeutic agent Internet Karyotype LIF gene Leukocytes Messenger RNA MicroRNAs Microspheres Molecular Profiling Monitor Morphology Motor Neurons Mus Names Neurons Notch Signaling Pathway Oligonucleotide Probes Online Systems Organ Ovary Population Process Regulator Genes Replacement Therapy Resources Slide Staining method Stains Stem cells Structure Supporting Cell Testing Tetracyclines Time Tissues Transcript Undifferentiated Up-Regulation Work base blastocyst cDNA Library cell bank cell type cholinergic embryonic stem cell gamma-Aminobutyric Acid mouse genome nerve stem cell overexpression pluripotency polysialyl neural cell adhesion molecule relating to nervous system research study self-renewal stem cell differentiation tool transcription factor transcriptome user-friendly

项目摘要

We have been developing tools and resources that make it possible to analyze a large number of genes in various experimental conditions. In our earlier work, we 1) constructed cDNA libraries from early mouse embryos and stem cells and generated a large number of expressed sequence tags (ESTs), 2) developed a glass-slide microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 44,000 unique mouse transcripts, 3) produced the web-based ANOVA-FDR software to provide user-friendly microarray data analysis, and 4) developed an algorithm and a fully-automated computational pipeline for transcript assembly from expressed sequences aligned to the mouse genome. In addition, we developed a comprehensive database and web browser of the binding sites of transcription factors (TFs) and cis-regulatory modules (CRMs) on the mouse genome. These resources and tools have then been applied to the systematic analysis of gene regulatory networks in mouse embryonic stem cells. Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. We have demonstrated that it is possible to analyze and identify downstream target genes by monitoring the global gene expression patterns of mouse ES cell lines, when a gene encoding a specific TF is manipulated so that the gene can be overexpressed or repressed. Previously, we generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. In further detail, we inferred an increased proportion of cells with neural progenitor marker PSA-NCAM after induction of several TFs. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In additional studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. We also explored the balance between seemingly antagonistic effects of RA on ESCs: differentiation and support of pluripotency. Although ESCs indeed differentiated in the presence of LIF after RA treatment, colonies of undifferentiated ESCs eventually emerged from these differentiated cells even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies after transfer to the standard ESC medium retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

我们一直在开发工具和资源，使其能够在各种实验条件下分析大量基因。在我们早期的工作中，我们1）构建了来自早期小鼠胚胎和干细胞的cDNA文库，并产生了大量的表达序列标签（EST），2）开发了一个载玻片微阵列平台，该平台包含原位合成的60-mer寡核苷酸探针，代表了大约44，000个独特的小鼠转录本，3）制作了基于网络的ANOVA-FDR软件，以提供用户友好的微阵列数据分析，和4）开发了一种算法和全自动计算管道，用于从与小鼠基因组比对的表达序列组装转录物。此外，我们开发了一个全面的数据库和网络浏览器的结合位点的转录因子（TF）和顺式调控模块（CRM）的小鼠基因组。这些资源和工具已被应用于小鼠胚胎干细胞基因调控网络的系统分析。小鼠胚胎干细胞（embryonic stem cells，ESCs）在体外可分化为多种细胞类型，为系统研究转录因子在细胞分化中的作用提供了理想的平台。我们已经证明，它是可能的，以分析和确定下游靶基因，通过监测全球基因表达模式的小鼠ES细胞系，当一个基因编码一个特定的TF被操纵，使该基因可以过表达或抑制。之前，我们生成并分析了137个TF诱导的小鼠ESC系。作为该“NIA小鼠ESC库”的延伸，我们产生并表征了48个额外的小鼠ESC系，其中每个系中的单个TF可以以强力霉素可控的方式诱导。与先前的ESC系一起，该库现在包括185个TF可操纵的ESC系（>所有小鼠TF的10%）。全局基因表达（转录组）分析显示，在小鼠ESC中诱导单个TF 48小时使其转录组向特定分化命运（例如，Myt 1 Isl 1和St 18表示神经谱系; Pitx 1、Pitx 2、Barhl 2和Lmx 1a表示中胚层谱系; Myb、Etv 2和Tbx 6表示白色血细胞; Pitx 1、Pitx 2和Dmrtc 2表示卵巢谱系）。这些数据还提供了每个TF的推断的靶基因和这些TF的可能功能的列表。这些结果证明了小鼠ESC系及其转录组数据对于理解细胞分化机制和TF功能的实用性。更详细地说，我们推断在诱导几种TF后，具有神经祖细胞标志物PSA-NCAM的细胞比例增加。我们确定Notch信号通路的早期激活是最有效的神经分化诱导剂的共同特征。通过Ascl 1、Smad 7、Nr 2f 1、Dlx 2、Dlx 4、Nr 2f 2、Barhl 2和Lhx 1诱导产生的大多数神经元样细胞是GABA阳性的，并表达GABA能神经元的其他标志物。以同样的方式，我们确定Lmx 1a和Nr 4a 2作为多巴胺能标记神经元的诱导物，Isl 1，Fezf 2和St 18作为胆碱能运动神经元的诱导物。诱导Ascl 1的时程实验显示，大多数神经特异性信使RNA（mRNA）和microRNA（miRNA）的早期上调。Ascl 1诱导的mRNA和miRNA组在Ascl 1靶中富集。在另外的研究中，使用微珠富集通过Ascl 1、Smad 7和Nr 2f 1诱导获得的细胞，产生基本上纯的神经元样细胞群，其表达谱与神经组织相似，并表达GABA能神经元的标志物。我们还探讨了RA对ESCs的看似拮抗作用之间的平衡：分化和支持多能性。尽管在RA处理后，在LIF存在下ESC确实分化，但即使在RA存在下，未分化的ESC的集落最终也从这些分化的细胞中出现。这些集落称为次级集落，由三种细胞类型组成：表达多能性基因（如Pou 5 f1、Sox 2和Nanog）的典型未分化ESC;表达Zscan 4的细胞;以及位于殖民地外围的内胚层样细胞。确认了所有8个测试的ESC系形成次级集落的能力。转移到标准ESC培养基后，来自次级集落的细胞保留了多能性，通过其强碱性磷酸酶（ALP）染色、典型的集落形态、基因表达谱、稳定的核型、在拟胚体形成测定中分化成所有三个胚层的能力以及注射到胚泡中后对嵌合体的成功贡献来判断。基于流式细胞术分析（FACS），次级集落中Zscan 4阳性细胞的比例高于标准ESC集落，这可以解释ESC抵抗RA的分化作用并形成未分化ESC的次级集落的能力。这一假设得到了细胞谱系追踪分析的支持，该分析显示次级集落中的大多数细胞是瞬时表达Zscan 4的细胞的后代。