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，使得在各种实验条件下分析大量基因成为可能。在我们的早期工作中，我们1）从早期的小鼠胚胎和干细胞中构造了cDNA库，并产生了大量表达的序列标签（ESTS），2）开发了一个玻璃扫描的微阵列平台，该平台包含含有原位合成的60-mer少物核苷酸探针，该探针大约是44,000个MIC分析的MIC分析，可用于3个典型的FRORMINES ANSOV-FDR-FDR DACAR-FDR DRACAR-FDR-FDR DRACR-FDR DRACR-FDR DRACR FRDR FRDR，3）和4）从与小鼠基因组相一致的表达序列的转录物组件开发了一种算法和完全自动化的计算管道。此外，我们开发了小鼠基因组上转录因子（TFS）和顺式调节模块（CIS调节模块（CRM）的结合位点的综合数据库和Web浏览器。然后将这些资源和工具应用于小鼠胚胎干细胞中基因调节网络的系统分析。小鼠胚胎干细胞（ESC）可以在体外分化为广泛的范围，并可能在体外分化，从而为细胞分化中转录因子（TFS）的作用提供了理想的平台。我们已经证明，当操纵编码特定TF的基因时，可以通过监测小鼠ES细胞系的整体基因表达模式来分析和识别下游靶基因，从而可以过表达或抑制该基因。以前，我们生成并分析了137个TF诱导的小鼠ESC系。作为此“ NIA鼠标ESC银行”的扩展，我们生成并表征了48个额外的鼠标ESC线，其中每行中的单个TF可以以强力控制的方式诱导。与先前的ESC线一起，该银行现在包括185个TF可操作的ESC线（>所有鼠标TF的10％）。全球基因表达（转录组）分析表明，小鼠ESC中单个TF的诱导持续48小时将其转录组转向特定的分化命运（例如，MyT1 ISL1的神经谱系，ST18； ST18;中胚层均由PITX1，PITX1，PITX2，BARHL2，BARHL2，和LMX1A和ORB乘MyBx22； PITX1，PITX2和DMRTC2）。这些数据还提供了每个TF的推断目标基因以及这些TF的可能功能的列表。结果证明了小鼠ESC线及其转录组数据的实用性，以了解细胞分化的机理和TF的功能。进一步详细地，我们推断出几种TF诱导后使用神经祖细胞标记物PSA-NCAM增加了细胞的比例增加。我们将Notch信号通路的早期激活视为大多数有效诱导者神经分化的共同特征。通过诱导ASCL1，SMAD7，NR2F1，DLX2，DLX4，NR2F2，BARHL2和LHX1产生的大多数神经元样细胞是GABA阳性，并且表达了其他GABA能神经元的标记。以同样的方式，我们将LMX1A和NR4A2鉴定为具有多巴胺能标记物的神经元的诱导剂，以及用于胆碱能运动神经元的ISL1，FEZF2和ST18。诱导ASCL1的时间课实验显示了大多数神经特异性的信剂RNA（mRNA）和microRNA（miRNA）的早期上调。 ASCL1诱导的mRNA和miRNA集合在ASCL1靶标中。在其他研究中，使用微珠诱导ASCL1，SMAD7和NR2F1获得的细胞富集导致基本上具有类似于神经组织的表达谱的神经元样细胞群，并具有类似于神经组织的表达谱和GABA能神经元的表达标记。我们还探讨了RA对ESC的看似拮抗作用之间的平衡：分化和对多能性的支持。尽管在RA处理后的LIF存在下确实有分化的ESC，但即使在RA存在的情况下，未分化的ESC的菌落也最终从这些分化的细胞中出现。这些菌落称为次生菌落，由三种细胞类型组成：表达多能基因的典型未分化的ESC，例如POU5F1，SOX2和Nanog；表达ZSCAN4的细胞;和内胚层状细胞位于菌落周围。在所有八个经过测试的ESC线路上，确认形成次要菌落的能力。转移到标准ESC培养基后次生菌落的细胞保留了多能性，这些细胞由其强大的碱性磷酸酶（ALP）染色，典型的菌落形态，基因表达谱，稳定的核型，稳定的核型，稳定的核型，可在胚胎身体形成鉴定中分化为所有三个细菌层的能力，并成功地贡献了对囊肿后进入爆破后的旋转。基于流式细胞仪分析（FACS），二级菌落中ZSCAN4阳性细胞的比例高于标准ESC菌落，这可以解释ESC的能力抵抗RA的分化作用，而是形成未分化的ESC的二级菌落。该假设得到了细胞训练的追踪分析的支持，这表明次生菌落中的大多数细胞是瞬时表达ZSCAN4的细胞的后代。