Project description:The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: in the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling downregulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-kB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a new mode of Cic regulation unrelated to the well-established RTK-Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.
Project description:Global phosphoproteomics can reveal signaling cascades in unprecedented detail. The SCF/KIT axis represents a vital hematopoietic receptor tyrosine kinase (RTK) system with gain-of-function mutations prompting the development of mastocytosis and leukemia. Here, we employed a label-free phosphoproteomics profiling strategy to provide the first comprehensive portrayal of SCF-triggered phosphosignaling. Employing untransformed skin-derived mast cells expressing vast amounts of KIT, we uncover around 10,500 class-I-phosphosites following ligand-induced KIT dimerization. Apart from STAT5, the MEK/ERK cascade (including upstream and downstream events) stood out as highly stimulatable by SCF compared to less impacted PI3K/Akt>p38>JNK, a pattern validated by time-resolved immunoblotting. Direct comparison between MEK/ERKÕs and PI3KÕs support of basic programs (anti-apoptosis, cell cycle progression) revealed equipotency with substantial redundancy between the modules. In functional outputs, ERK showed dominance over PI3K regarding cytokine stimulation (TNF-a, IL-8, LIF, OSM) and was the only contributor in the induction of immediate early genes (Fos, JunB, Egr1). Our proteome-wide screen also found Å1,000 phosphosites currently undescribed in databases, pertaining to multiple protein classes. Among candidates, capicua (and components of its network) experienced massive phosphorylation by SCF. Perturbation with capicua by RNA interference unveiled its function as a potent KIT repressor, whose deregulation may contribute to the development of mastocytosis. We demonstrate the utility of an unbiased approach to uncover single protein entities and entire networks activated by RTKs, that can be surveyed experimentally. This rich resource enhances our understanding of RTK-elicited signaling networks and can serve as a benchmark to identify pathological processes, e.g. under circumstances of KIT deregulation.
Project description:Analysis of cerebella from Capicua (Cic) mutant mice and wild-type controls at 28 days of age (P28). Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). The transcriptional repressor Cic binds directly to Atxn1 and plays a key role in SCA1 pathogenesis. Two isoforms of Cic, long (Cic-L) and short (Cic-S), are transcribed from alternative promoters. Using embryonic stem cells in which the Cic locus was targeted by an insertion of a genetrap cassette between exon 1 of the Cic-L isoform and exon 1 of the Cic-S isoform, we generated mice that carried this allele and backcrossed these onto a Swiss Webster (CD-1) strain for >6 generations. The resulting Cic-L-/- mice completely lack the Cic-L isoform with ~10% of Cic-S remaining. These data were used to compare with previous microarray data to determine the Cic-depedent pathogenic mechanisms in SCA1. Total RNA from cerebella of wild-type (n=4) and Capicua mutant mice (n=4) at 28 days of age was prepared and labeled according the manufacturer's protocols for the Affymetrix Mouse Gene 1.0 ST Array.
Project description:Background: Seizures can present at any time before or after the diagnosis of a glioma. Roughly, 25-30 % of glioblastoma (GBM) patients initially present with seizures, and an additional 30 % develop seizures during the course of the disease. Early studies failed to show an effect of general administration of anti-epileptic drugs for glioblastoma patients, since they were unable to stratify patients into high- or low- risk seizure groups. Methods: 112 patients, who underwent surgery for a GBM, were included. Genome- wide DNA methylation profiling was performed, before methylation subclasses and copy number changes inferred from methylation data were correlated with clinical characteristics. Independently, global gene expression was analyzed in GBM methylation subclasses from TCGA datasets (n=68). Results: Receptor tyrosine Kinase (RTK) II GBM showed a significantly higher incidence of seizures than RTK I and mesenchymal (MES) GBM (p<0.01). Accordingly, RNA expression datasets revealed an upregulation of genes involved in neurotransmitter synapses and vesicle transport in RTK II glioblastomas. In a multivariate analysis, temporal location (p=0.01, OR 6.35) and RTK II (p=0.03, OR 5.56) were most predictive for preoperative seizures. During postoperative follow-up, only RTK II remained significantly associated with the development of seizures (p<0.01, OR 8.23). Consequently, the need for antiepileptic medication and its increase due to treatment failure was highly associated with the RTK II methylation subclass (p<0.01). Conclusion: Our study shows a strong correlation of RTK II glioblastomas with preoperative and long-term seizures. These results underline the benefit of molecular glioblastoma profiling with important implications for postoperative seizure control.
Project description:We have measured by ChIP-seq the binding of Capicua transcriptional repressor (Cic) in Drosophila melanogaster embryos at early nuclear cycle 14. We compared binding in response to ERK activation, utilizing optogenetics.
Project description:Receptor tyrosine kinase pathway signalings plays a central role in the growth and progression of glioblastoma, a highly aggressive group of brain tumors. We recently reported that miR-218 repression, an essentially uniform feature of human GBM, directly promotes RTK hyperactivation by increasing the expression of key positive signaling effectors, including EGFR, PLCr1, PIK3CA and ARAF. However, enhanced RTK signaling is known to activate compensatory inhibitory feedback mechanisms in both normal and cancer cells. We demonstrate here that miR-218 repression in GBM cells also increases the abundance of additional up stream and downstream signaling mediators, including PDGFRa, RSK2, and S6K1, which collectively funciton to alleviate inhibitory RTK feedback regulation. In turn, RTK signaling suppresses miR-218 expression via STAT3, which binds to the miR-218 locus, along with BCLAF1, to repress its expression. These data identify novel interacting feedback loops by which miR-218 repression promotes increased RTK signaling in high-grade gliomas.