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SUMMARY Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

α 2 -adrenoceptors are essential presynaptic regulators of norepinephrine release from sympathetic nerves. Previous studies in mice with targeted deletions in the 3 α 2 -adrenoceptor genes have indicated that these receptors are essential for embryonic development. In the present study, we searched for the α 2 -adrenoceptor subtype(s) involved in placental development and its molecular mechanism using mice carrying targeted deletions in α 2 -adrenoceptor genes. Congenic α 2B -adrenoceptor–deficient mice ( Adra2b −/− ) developed a defect in fetal and maternal vessel formation in the placenta labyrinth at embryonic day 10.5. This defect was accompanied by reduced endothelial cell proliferation and decreased extracellular signal-regulated kinase 1/2 phosphorylation levels in Adra2b −/− as compared with Adra2b +/+ placentae. Microarray analysis of wild-type and mutant placentae (maternal genotype Adra2b +/− ) revealed 179 genes, which were significantly up- or downregulated >1.5-fold in α 2B -deficient placentae. The type 1 receptor for vascular endothelial growth factor (Flt1), which is coexpressed with α 2B -adrenoceptors in spongiotrophoblast and giant cells of the placenta, was found to be 2.3-fold upregulated in α 2B -deficient placentae. Neutralization of Flt1 and its soluble splice variant sFlt1 by a specific antibody in vivo prevented the vascular defect in α 2B -deficient placentae at embryonic day 10.5. Thus, α 2B -adrenoceptors are essential to suppress antiangiogenic (s)Flt1 in spongiotrophoblasts to control the coordinated formation of a vascular labyrinth of fetal and maternal blood vessels in the murine placenta during development.

Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe -/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe -/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe -/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH.

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-alpha (ERalpha), resulting in subsequent clearance of ERalpha protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERalpha positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERalpha mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERalpha expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERalpha from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERalpha, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.

Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefbeta transcription is directly regulated by light while transcription of tefalpha is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo.

Non-coding RNA profiles in cancer are largely unknown which greatly impedes the discovery of functionally important ncRNAs in tumorigenesis as well as the generation of genome-wide libraries. Here, we define the ncRNA expression landscape of lung, breast and liver cancer as well as normal tissue from the respective organs in a large set of primary patient samples (N=150). These samples were carefully selected to have a long patient follow-up and complete clinical datasets to allow an in-depth analysis. We provide the first comprehensive map of 17000+ long ncRNAs in a broad range of human tumor and normal tissues and discovered hundreds of new ncRNAs associated with three major tumor entities. Importantly, we uncovered that ncRNA profiles are significantly more specific to the tissue of origin than patterns of protein-coding mRNAs. Also, the significant ncRNA signatures in this study demonstrate specific ncRNA patterns that are unlikely to be transcriptional background but rather the result of concerted regulation. To mimic the response to cytotoxic chemotherapy, we have also treated lung and liver cancer cells with the DNA damaging agents Cisplatin and Etoposide and found significant deregulation of long ncRNAs - reversing in part the differences seen between normal and malignant lung tissue. Importantly, one of the Cisplatin-regulated ncRNAs interacts with DNA repair factors linking it immediately to the DNA damage response. To elucidate the molecular functions of the novel ncRNAs, we used RNA affinity purification to identify the protein interaction partners. In addition, we create human knockout cells for lncRNAs using ZFN and TALEN technology to integrate RNA destabilizing elements into the human genome which yields more than 1000-fold lncRNA silencing. Our so far largest ncRNA expression map is also exploited in another way: We generate an siRNA library specifically targeting over 600 tumor-associated ncRNAs based on our profiling landscape. This comprehensive but focused library will elucidate the role of ncRNAs in tumorigenesis, viability, apoptosis and the DNA damage response. In summary, we provide the first global comprehensive map of long ncRNA expression in a broad range of human tumor and normal tissue samples and discovered many new lncRNAs associated with cancer as well as tissue-, histology- and prognosis-specific ncRNA signatures. Citation Format: Maria Polycarpou-Schwarz, Tony Gutschner, Monika Hämmerle, Anna Roth, Ashish Goyal, Stefanie Grund, Catherina Hildenbrand, Arne Warth, Thomas Longerich, Sebastian Aulmann, Joachim Rom, Michael Meister, Thomas Muley, Heike Zabeck, Sabine Schmidt, Tomi Ivacevic, Vladimir Benes, Kai Breuhahn, Philipp Schnabel, Peter Sinn, Hans Hoffmann, Peter Schirmacher, Sven Diederichs. The non-coding RNA landscape of lung, liver and breast cancer reveals novel tumor-associated ncRNAs, concerted regulation and unexpected tissue specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2013-1824