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.
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