During this reporting period the Laboratory of Genetics and Physiology has made progress and elucidated mechanisms by which cytokines control mammary epithelium, hematopoietic lineages and liver through the transcription factors STAT1 and STAT5. In addition, LGP has generated for the first time mice in which the gene encoding the transcription factor STAT1 is bracketed by loxP sites and can therefore be deleted in a cell-specific and conditional fashion. Mammary cancer Studies from our laboratory have demonstrated for the first time that the transcription factor STAT1 is a tumor suppressor in mammary epithelium. Type I and type II classes of interferons (IFNs) signal through the JAK/STAT1 pathway and are known to be important in adaptive and innate immune responses and in protection against tumors. Although STAT1 is widely considered a tumor suppressor, it remains unclear, however, if this function occurs in tumor cells (cell autonomous) or if STAT1 acts primarily through immune cells. The question whether STAT1 has a cell autonomous role in mammary tumor formation was addressed in a mouse model of ERBB2/neu-induced breast cancer in the absence and presence of STAT1. For this purpose, mice that carry floxed Stat1 alleles, which permit cell-specific removal of STAT1, were generated. To induce tumors only in mammary cells lacking STAT1, Stat1 floxed mice were crossed with transgenic mice that express cre recombinase and the neu oncogene under the mouse mammary tumor virus LTR (Stat1fl/fl NIC). Stat1 was effectively deleted in mammary epithelium of virgin Stat1fl/fl NIC females. Time-to-tumor onset was significantly shorter in Stat1fl/fl NIC females than in WT NIC. The median time-to-tumor onset in the Stat1fl/fl NIC mice was 49 weeks, whereas it was 62 weeks in the WT NIC mice. These results demonstrate that STAT1 in mammary epithelial cells functions as a tumor suppressor. The Stat1 floxed allele we have generated is also a unique resource to determine the cellular targets of IFNs and STAT1 action, which should aid our understanding and appreciation of these pathways. These mice have been distributed to more than a dozen laboratories already. Liver metabolism and hepatocellular carcinoma LGP scientists have established that the transcription factor STAT5 is a context-specific tumor suppressor. Although the cytokine-inducible transcription factor signal transducer and activator of transcription 5 (STAT5) promotes proliferation of a wide range of cell types, there are cell-specific and context-specific cases in which loss of STAT5 results in enhanced cell proliferation. We have reported that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15(INK4B) and p21(CIP1). We have demonstrated that growth hormone, through the transcription factor STAT5, enhances expression of the Cdkn2b (cyclin-dependent kinase inhibitor 2B) gene and that STAT5A binds to interferon-gamma-activated sequence sites within the promoter. In this study we have also established that STAT5, like in MEFs, activates expression of the Cdkn2b gene in liver tissue. Loss of STAT5 led to diminished p15(INK4B) and increased hepatocyte proliferation. This study for the first time demonstrated that cytokines, through STAT5, induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context-specific role of STAT5 as tumor suppressor. Cytokines and hematopoietic stem cells It has been established that cytokines control the fitness of hematopoietic stem cells, which in turn has direct implications on the development and treatment of leukemias. We have performed large-scale gene expression profiling and identified a the ccn3/nov gene as a prominent target of STAT5. Notably we were able to demonstrate that this gene is activated by interleukin 3 (IL-3) in HSCs. The Ccn3 gene is of particular interest as it has a defined role in the biology of HSCs and it is highly expressed in leukemias. In a collaborative research program LGP scientists have contributed to the finding that STAT5 has oncogenic properties in hematopoietic tumors carrying ETV6-LYN translocations. Cre transgenic mice More than a decade ago this laboratory has generated various transgenic mouse strains that express Cre recombinase in mammary epithelium and in a limited set of other cell types. Since then these mice have been used successfully to to target gene deletions by more than one hundred investigators. We had some evidence that female mice from one of these transgenic mouse lines had problems raising their litters, probably due to some impairment in mammary development and/or function. Since this finding has potentially important implications in the interpretation of gene knock-out studies, we performed a detailed study on the lactation performance and mammary development in these mice.

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Lee, Hye Kyung; Willi, Michaela; Wang, Chaochen et al. (2017) Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice. Nucleic Acids Res 45:4606-4618
Willi, M; Yoo, K H; Reinisch, F et al. (2017) Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells. Nat Commun 8:16069
Shin, Ha Youn; Wang, Chaochen; Lee, Hye Kyung et al. (2017) CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat Commun 8:15464
Willi, Michaela; Yoo, Kyung Hyun; Wang, Chaochen et al. (2016) Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation. Nucleic Acids Res 44:10277-10291
Shin, Ha Youn; Willi, Michaela; HyunYoo, Kyung et al. (2016) Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat Genet 48:904-911
Hosui, Atsushi; Tatsumi, Tomohide; Hikita, Hayato et al. (2016) STAT5 plays a crucial role in hepatic lipid metabolism through regulation of CD36 expression. Hepatol Res :
Metser, Gil; Shin, Ha Youn; Wang, Chaochen et al. (2016) An autoregulatory enhancer controls mammary-specific STAT5 functions. Nucleic Acids Res 44:1052-63
Lee, Jongwon; Seong, Semun; Kim, Jung Ha et al. (2016) STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis. Sci Rep 6:30977
Müller, T A; Grundler, R; Istvanffy, R et al. (2016) Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype. Leukemia 30:1725-33
Yoo, Kyung Hyun; Oh, Sumin; Kang, Keunsoo et al. (2016) Histone Demethylase KDM6A Controls the Mammary Luminal Lineage through Enzyme-Independent Mechanisms. Mol Cell Biol 36:2108-20

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