Genetic Approaches To Understanding Organ Development and Function

了解器官发育和功能的遗传学方法

• 批准号: 8741539
• 负责人: Lothar Hennighausen
• 金额: $ 137.83万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 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/8741539
• 关键词: Acute Myelocytic Leukemia; Address; Alleles; Allergic Disease; Alveolar; B-Lymphocytes; BCL2L11 gene; Basophils; Binding; Binding Sites; Biological; Biology; CD4 Positive T Lymphocytes; Cell Lineage; Cell model; Cell physiology; Cells; ChIP-seq; Chromatin; Contact hypersensitivity; Cytokine Signaling; DNA Sequence; Data Set; Databases; Dendritic Cells; Dendritic cell activation; Development; Dinitrofluorobenzene; Disease; Enzymes; Epithelial; Family; Family member; Female; Fluorescein-5-isothiocyanate; Gene Expression Profile; Gene Targeting; Genes; Genetic; Genetic Programming; Genetic Transcription; Genome; Genomics; Gluconeogenesis; Goals; Haptens; Hematopoiesis; Hematopoietic Neoplasms; Hematopoietic System; Hepatic; Hepatocyte; Histologic; Human; Immigration; In Vitro; Individual; Inflammatory Response; Interferon Type II; Investigation; Knockout Mice; Laboratories; Lactation; Langerhans cell; Link; Liver; Malignant neoplasm of liver; Mammary gland; Mediating; Messenger RNA; Meta-Analysis; Metabolic Diseases; Metformin; MicroRNAs; Mind; Minor; Modeling; Molecular; Mus; NCOA2 gene; Organ; Pathway interactions; Pattern; Phosphorylation; Physiological; Physiology; Pregnancy; Primary carcinoma of the liver cells; Process; Protein Tyrosine Kinase; Pyruvate Dehydrogenase Complex; Regulation; Reporting; Research; Resources; Role; STAT1 gene; STAT3 gene; STAT4 gene; STAT5A gene; STAT5B gene; STAT6 gene; Scientist; Signal Pathway; Signal Transduction; Site; Specificity; Staging; Stem cells; Stimulus; T-Lymphocyte; Therapeutic; Tissues; Tumor Suppressor Proteins; Undifferentiated; cell type; chromatin immunoprecipitation; combat; comparative; cytokine; design; eosinophil; genome-wide; human TSLP protein; in vivo; insight; leukemic stem cell; liver metabolism; macrophage; mammary epithelium; mammary gland development; member; monocyte; mouse model; novel; novel therapeutic intervention; peptide hormone; pro-apoptotic protein; programs; promoter; pyruvate dehydrogenase kinase 4; response; tool; transcription factor

During this reporting period the Laboratory of Genetics and Physiology has made progress and elucidated mechanisms by which cytokines control mammary development during pregnancy, normal liver physiology and the development of liver cancer and hematopoietic malignancies through the transcription factors STAT1 and STAT5. In addition, we investigated the role of cytokine-induced miRNA genes in mammary gland development. Comparative genome-wide binding of STATs identified cis-regulatory modules Using public databases we have performed a comprehensive meta-analysis of STAT genomic binding patterns and identified cell-specific cis-regulatory modules. Although the seven STAT members recognize similar, if not identical, DNA sequence motifs in vitro, they execute cell- and context-specific functions. Yet, cell-specific gene expression patterns are obtained despite different cells being exposed in vivo to similar cytokines. Direct STAT binding to cognate genomic targets will, at least in part, effect cytokine stimuli. With this in mind, new and critical insight into common and cell-specific functions of STATs could come from genome-wide STAT occupancy data sets. However, it is not clear to what extent different members of the STAT family share genetic targets. STAT binding to the canonical GAS (gamma interferon-activated sequence) motif (TTCnnnGAA), the extent of cell specificity and the influence of STAT concentration on their ability to occupy genomic sites are poorly understood. Large-scale chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) studies have explored in vivo binding of five different STATs in a number of different cell types exposed to several cytokines. We have now comparatively reanalyzed this resource of 29 data sets and gained insight into the complexity of common and selective STAT binding patterns that are unique to, as well as shared between, different cell lineages. We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. Even for the top 1,000 highly enriched STAT binding sites, 15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells 90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors. Mammary development Integrating mouse genetics with large-scale genomic analyses led to the discovery that the sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on the concentration of STAT5. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts mammary epithelial development during pregnancy. The presence of only a single Stat5a or Stat5b allele was sufficient for the establishment of histologically undifferentiated alveolar units and two alleles permitted the execution of a differentiation program similar to that found with all four alleles. While one copy of Stat5 induced limited expression of target genes, two copies activated a lactation-like gene signature. Using ChIP-seq analyses on intact tissue under physiological conditions, we found that highly regulated genes were bound by STAT5 in their promoter proximal regions whereas upstream binding had minor biological consequences. Remarkably, 80% of the genes bound by STAT5 in vivo were not under STAT5 control. Liver metabolism and hepatocellular carcinoma Using mouse genetics tools LGP scientists have shown previously that the transcription factor STAT5 controls the physiology of liver tissue and that loss of STAT5 results in hepatosteatosis and hepatocellular carcinoma (HCC). During this and the previous reporting period we discovered that the liver-specific tumor suppressor STAT5 controls expression of the ROS generating enzyme NOX4 and the pro-apoptotic proteins PUMA and BIM. These results demonstrate that STAT5 harnesses several distinct signaling pathways in liver to exert tumor suppressor functions. GH not only controls the physiology of hepatocytes but aberrant GH signaling has been linked to metabolic disorders. In a collaborative study we discovered that metformin might provide a novel therapeutic approach for the treatment of hepatic metabolic disorders induced by aberrant GH signaling. Inhibition of the pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 4 (PDK4) conserves substrates for gluconeogenesis and we were able to demonstrate that the administration of GH in vivo increased PDK4 expression via a pathway dependent on STAT5 phosphorylation. Our findings provide evidence for a novel pathway for the regulation of hepatic PDK4 expression and metabolites by GH and a potential therapeutic approach for the treatment of hepatic metabolic disorders. Hematopoiesis In the past we have provided insight into mechanisms by which cytokines control various aspects of hematopoiesis through the transcription factor STAT5. These studies revolved around conditional gene knock-out mice generated in LGP. LGP scientists have now participated in a study demonstrating that STAT5 is essential for specific cytokine responses of dendritic cells (DCs) but not for their overall development and function. The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has been implicated in allergic disease in humans and in type 2 inflammatory responses in mice. Several cell types have been shown to be TSLP-responsive, including DCs, Langerhans cells, CD4 T cells, B cells, basophils, eosinophils, and monocyte/macrophages. However, several lines of evidence have indicated that DCs are the primary in vivo target of TSLP action. Using mice with a DC-specific deletion of STAT5, we showed that STAT5 is required for TSLP-dependent DC activation. Furthermore, these mice showed a markedly reduced response in a TSLP-dependent model of FITC-mediated contact hypersensitivity (CHS). Importantly, these mice responded normally to the TSLP-independent hapten dinitrofluorobenzene (DNFB). These results suggest that Stat5 is required for TSLP-dependent DC activation and migration in type-2 CHS responses. In another collaborative study it was shown that STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2. miRNAs and mammary physiology Micro RNAs (miRNAs) are believed to be important post transcriptional regulators of messenger RNAs that modulate the physiology of cells. LGP scientists have identified miRNAs that are under cytokine-STAT5 control, suggesting a role of miRNAs in those cells that depend on the presence of STAT5. To address the relevance of specific miRNAs regulated by STAT5 we performed studies to delete specific STAT5 dependent miRNA genes. In the current reporting period we have deleted a cluster composed of several miRNAs (miR-17-92) from mammary stem cells in mice and explored the consequences on mammary development and function. Unexpectedly, mammary tissue devoid of these miRNAs underwent normal developed during pregnancy and mice were able to lactate. Thus, at least this miRNA cluster is not required for normal mammary physiology. We are in the process of evaluating the role of additional miRNAs in the mammary gland and liver.

在这一报告期间，遗传学和生理学实验室已经取得了进步和阐明的机制，细胞因子在怀孕期间控制乳腺发育，正常肝脏生理以及通过转录因子STAT1和STAT5的肝癌和造血性恶性肿瘤的发展。此外，我们研究了细胞因子诱导的miRNA基因在乳腺发育中的作用。 鉴定出顺式调节模块的统计数据的比较全基因组结合 使用公共数据库，我们对Stat基因组结合模式进行了全面的荟萃分析，并确定了细胞特异性的顺式调节模块。尽管七个Stat成员在体外识别相似的DNA序列基序，但它们会执行细胞和上下文特异性功能。然而，尽管在体内暴露于相似的细胞因子中，但仍获得了细胞特异性基因表达模式。与同源基因组靶标的直接统计结合至少部分影响细胞因子刺激。考虑到这一点，对统计数据的常见和细胞特异性功能的新和批判性洞察力可能来自全基因组统计数据占数据集。但是，尚不清楚STAT家族的不同成员在多大程度上具有遗传靶标。统计与规范气体（伽马干扰素激活序列）基序（TTCNNNGAA）的​​统计结合，细胞特异性的程度以及Stat浓度对其占据基因组位点能力的影响。大规模的染色质免疫沉淀，然后进行高吞吐量测序（CHIP-SEQ）研究，在暴露于几种细胞因子的许多不同细胞类型中探索了五个不同统计数据的体内结合。现在，我们已经相对重新分析了29个数据集的资源，并深入了解了共同和选择性统计结合模式的复杂性，这些模式是独特的，并且在不同的细胞谱系之间共享。 我们确定统计数据的基因组结合能力主要由细胞类型定义，并且在较小程度上由单个家庭成员定义。即使对于前1,000个高度富集的统计结合位点，小鼠雌性肝脏中15％的STAT5结合位点在不同细胞类型的其他统计数据中共享，而在T细胞中，STAT3，STAT3，STAT4和STAT6在T细胞中共有90％的STAT5结合位点。此外，我们确定了116个顺式调节模块（CRM），这些模块在定义常见JAK-STAT签名的细胞类型的所有STAT成员中都识别出来。最后，在肝脏STAT5结合中，与细胞特异性转录因子HNF4A，FOXA1和FOXA2的结合显着重合，并且与细胞类型特异性基因转录有关。我们的结果表明，Stats的基因组结合主要取决于细胞类型，并且进一步的特异性部分是通过并置的细胞特异性转录因子并置的。 乳腺发展 将小鼠遗传学与大规模基因组分析的整合导致发现，发现妊娠期间小鼠乳腺上皮细胞中遗传程序的顺序激活取决于STAT5的浓度。我们已经使用了小鼠遗传学和全基因组分析来确定STAT5A和Stat5b浓度的改变如何影响妊娠期间乳腺上皮发育。仅存在单个STAT5A或STAT5B等位基因就足以建立组织学上未分化的肺泡单位，而两个等位基因允许执行与所有四个等位基因相似的分化程序。尽管STAT5的一份副本诱导靶基因表达有限，但两个副本激活了哺乳样基因的特征。使用在生理条件下对完整组织的芯片序列分析，我们发现高度调节的基因在其启动子近端区域受到STAT5的结合，而上游结合则具有较小的生物学后果。值得注意的是，由STAT5在体内绑定的基因中有80％不受STAT5对照。 肝代谢和肝细胞癌 使用小鼠遗传学工具LGP科学家先前已经表明，转录因子STAT5控制着肝组织的生理学，而STAT5的丧失导致肝脏和肝细胞癌（HCC）导致肝脏组织。在此期间和上一个报告期间，我们发现肝脏特异性肿瘤抑制剂STAT5控制ROS产生酶NOX4的表达以及促凋亡蛋白PUMA和BIM。这些结果表明，STAT5利用肝脏中的几种不同的信号通路来发挥肿瘤抑制功能。 GH不仅控制肝细胞的生理，而且异常的GH信号传导与代谢疾病有关。在一项合作研究中，我们发现二甲双胍可能提供一种新型的治疗方法，用于治疗异常GH信号引起的肝代谢疾病。丙酮酸脱氢酶激酶4（PDK4）对丙酮酸脱氢酶复合物（PDC）的抑制作用抑制糖异生的底物，我们能够证明，在体内GH的施用通过依赖于STAT5磷酸化的途径来增加PDK4的pDK4表达。我们的发现为通过GH调节肝PDK4表达和代谢物调节的新途径提供了证据，以及用于治疗肝代谢疾病的潜在治疗方法。 造血 过去，我们提供了对细胞因子通过转录因子Stat5控制各个方面的机制的见解。这些研究围绕着LGP产生的条件基因敲除小鼠。 LGP科学家现在已经参与了一项研究，该研究表明，STAT5对于树突状细胞（DC）的特定细胞因子反应至关重要，但对于它们的整体发育和功能而不是必不可少。上皮衍生的细胞因子胸腺基质淋巴结蛋白（TSLP）已与小鼠的人类和2型炎症反应中的过敏性疾病有关。已显示几种细胞类型具有TSLP响应性，包括DC，Langerhans细胞，CD4 T细胞，B细胞，嗜碱性粒细胞，嗜酸性粒细胞和单核细胞/巨噬细胞。但是，有几条证据表明DC是TSLP作用的主要体内目标。使用具有STAT5的DC特异性缺失的小鼠，我们表明STAT5是TSLP依赖性DC激活所必需的。此外，这些小鼠在FITC介导的接触超敏反应（CHS）的TSLP依赖性模型中显示出明显降低的反应。重要的是，这些小鼠正常反应于TSLP独立于二硝基氟苯（DNFB）。这些结果表明，依赖TSLP的DC激活和2型CHS响应中的迁移需要STAT5。在另一项协作研究中，表明STAT5对于维持MOZ-TIF2诱导的急性粒细胞性白血病中的白血病干细胞至关重要。 miRNA和乳腺生理学 据信微RNA（miRNA）是使细胞生理学调节的信使RNA的转录后调节剂。 LGP科学家已经确定了受细胞因子-STAT5控制的miRNA，这表明miRNA在依赖于STAT5存在的细胞中起作用。为了解决由STAT5调节的特定miRNA的相关性，我们进行了研究以删除特定的STAT5依赖miRNA基因。在当前的报告期间，我们删除了一个由小鼠乳腺干细胞的几个miRNA（miR-17-92）组成的簇，并探讨了对乳腺发育和功能的后果。出乎意料的是，没有这些miRNA在怀孕期间发生正常的乳腺组织，小鼠能够乳酸。因此，至少这种miRNA簇不需要正常的乳腺生理。我们正在评估其他miRNA在乳腺和肝脏中的作用。