Dysregulation of liver X receptor ␣ (LXR␣) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXR␣ target gene selectivity is achieved by modulation of LXR␣ phosphorylation. Under basal conditions, LXR␣ is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXR␣ S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXR␣ S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXR␣-responsive genes.

We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan À/À cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.

SUMMARYMicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila β-catenin) expression and activity by directly targeting the 3ʹ-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad. MATERIALS AND METHODS Cell culture and high-throughput screen (HTS)For the HTS, the Wg pathway was activated in Drosophila Clone 8 (Cl8) and S2R+ cells [grown as described by DasGupta et al. (DasGupta et al., 2005)] by introducing Axin double-stranded RNA (dsRNA), which resulted in a robust, ligand-independent activation of the Wg-responsive dTF12 reporter (DasGupta et al., 2005) (Fig. 1A). We screened a library of miRNA expression constructs [UAS-dsRED-pri-miR (Silver et al., 2007)] that consisted of 75 previously screened pri-miR constructs (Silver et al., 2007) plus 115 as yet unscreened pri-miR plasmids for their ability to suppress dTF12 activity downstream of the DC in this transcriptionally sensitized background. A total of 190 screen-ready plasmids were plated using a Janus MDT automated workstation (Perkin Elmer) in 5 µl aliquots as quadruplicates arranged in a quadrant on a set of three 384-well plates. Several quadrants of four replica wells were left empty for the addition of assay-specific controls. Axin dsRNA was generated using the Megascript kit (Applied Biosystems) using the following primers (5Ј-3Ј): forward TAATACGACTCACTATAGGGagaccaaacgccgcaccgctcgcc and reverse TAATACGACTCACTATAGGGagacaaaagccggtcgcccgtac (capital letters denote priming regions for T7 RNA polymerase).Cells were suspended at 20,000 cells/well for S2/S2R+ and 40,000 cells/well for Cl8. The dTF12-luciferase (TOP12-Ffl) reporter and Pol IIIRenilla luciferase (PolIII-RL) were utilized as described (DasGupta et al., 2005), with the addition of 0.01 µg actin-GAL4 and 0.1 µg Axin dsRNA, and transfected using the Effectene kit (Qiagen). Cells were incubated posttransfection for 5 days and luciferase levels assessed using the Promega Dual-Glo kit (Promega).For screen data analysis, Firefly luciferase activity values were normalized to those of Renilla luciferase for each...

The recent development of high-throughput sequencing technologies and the availability of whole genome sequences of a variety of living organisms, including that of humans, have led to an enormous push in the quest for a comprehensive inquiry for the function of each and every gene discovered in different model organisms. A major conclusion from the sequencing projects was that while forward genetics had been extremely successful in identifying key genes/components of many biological processes, such as signal transduction cascades, the function(s) of the majority of genes in the genome remains a mystery. In this article, we discuss the use of a variety of high-throughput postgenomic tools, including functional genomics, proteomics, and chemical genetics that are being implemented in an exhaustive molecular dissection of a key evolutionarily conserved signal transduction pathway, namely the Wnt/wingless (wg) pathway and its associated signaling network.