Systematic analysis of gene regulatory networks

我们一直在开发工具和资源，使在各种实验条件下分析大量基因成为可能。在我们早期的工作中，我们1)构建了小鼠早期胚胎和干细胞的cDNA文库，并生成了大量的表达序列标签(EST)；2)开发了一个玻片微阵列平台，其中包含了原位合成的60聚体寡核苷酸探针，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软件来提供用户友好的微阵列数据分析；以及4)开发了一种算法和全自动计算流水线来组装与小鼠基因组配对的表达序列。此外，我们还开发了一个完整的小鼠基因组转录因子(TF)和顺式调控模块(CRM)结合位点的数据库和网络浏览器。这些资源和工具随后被应用于系统分析小鼠胚胎干细胞的基因调控网络。 小鼠胚胎干细胞(ESCs)在体外可以分化为多种类型的细胞，为系统研究转录因子在细胞分化中的作用提供了一个理想的平台。我们已经证明，当编码特定TF的基因被操纵以使该基因能够过度表达或抑制时，通过监测小鼠ES细胞系的全局基因表达模式来分析和识别下游靶基因是可能的。在此之前，我们构建并分析了137个Tf诱导的小鼠胚胎干细胞系。作为这个“NIA小鼠ESC库”的扩展，我们另外产生并鉴定了48个小鼠ESC系，其中每个系中的单个TF可以以多西环素可控的方式被诱导。连同以前的ESC线，该库现在包括185条可操控的ESC线(占所有鼠标TF的10%)。全球基因表达谱(转录组)显示，单独的转录因子在小鼠ESCs中诱导48小时后，其转录本朝着特定的分化命运转变(例如，Myt1 Isl1和St18的神经系；Pitx1、Pitx2、Barhl2和Lmx1a的中胚层系；Myb、ETV2和Tbx6的白细胞；以及Pitx1、Pitx2和Dmrtc2的卵巢)。这些数据还提供了推测的每个转铁蛋白的靶基因和这些转铁蛋白的可能功能的列表。这些结果表明，小鼠胚胎干细胞系及其转录组数据对于了解细胞分化的机制和转录因子的功能是有用的。 更详细地，我们推测在几个TF诱导后，带有神经前体标记PSA-NCAM的细胞比例增加。我们发现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。Ascl1、Smad7、Nr2f1、Dlx2、Dlx4、Nr2f2、Barhl2和Lhx1诱导产生的神经元样细胞多为GABA阳性，表达GABA能神经元的其他标志。同样，我们发现Lmx1a和Nr4a2是带有多巴胺能标记的神经元的诱导剂，Isl1、Fezf2和St18是胆碱能运动神经元的诱导剂。Ascl1诱导的时程实验显示，大多数神经特异性信使RNA(MRNAs)和microRNAs(MiRNAs)早期上调。Ascl1诱导的多组mRNAs和miRNAs在Ascl1靶标中得到丰富。在其他研究中，用微球对Ascl1、Smad7和Nr2f1诱导获得的细胞进行浓缩，得到了基本上纯净的神经元样细胞群，其表达谱类似于神经组织，并表达了GABA能神经元的标志。 我们还探索了RA对ESCs的表面拮抗作用之间的平衡：分化和支持多能性。虽然RA治疗后ESCs确实在LIF存在的情况下分化，但即使在RA存在的情况下，未分化的ESCs最终也会从这些分化的细胞中分化出来。这些克隆被称为次级克隆，由三种细胞类型组成：表达Pou5f1、Sox2和Nanog等多潜能基因的典型未分化ESCs；表达Zscan4的细胞；以及位于克隆外围的内胚层样细胞。所有测试的8个ESC品系都证实了形成次级克隆的能力。将二次集落转移到标准的ESC培养液中后，从细胞的碱性磷酸酶(ALP)染色、典型的集落形态、基因表达谱、稳定的核型、在类胚体形成试验中分化为所有三个胚层的能力以及注射到囊胚后成功地形成嵌合体来判断，这些细胞保持了多能性。流式细胞仪分析显示，次级集落中Zscan4阳性细胞的比例高于标准ESC集落，这可能解释了ESCs抵抗RA的分化作用而形成未分化ESCs的次级克隆的能力。这一假设得到了细胞谱系追踪分析的支持，该分析表明，次级克隆中的大多数细胞是瞬时表达Zscan4的细胞的后代。

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.