Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase

The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer

Phosphorylation of the Drosophila melanogaster RNA–Binding Protein HOW by MAPK/ERK Enhances Its Dimerization and Activity

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA–binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW–mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles

Phosphorylation of groucho mediates RTK feedback inhibition and prolonged pathway target gene expression

[Background]: Signaling by receptor tyrosine kinase (RTK) pathways plays fundamental roles in processes of cell-fate determination, often through the induction of specific transcriptional responses. Yet it is not fully understood how continuous target gene expression, required for irreversible cell-fate specification, is preserved after RTK signaling has ended. Here we address this question using the Drosophila embryo, a model system that has been instrumental in elucidating the developmental functions of RTK signal transduction. [Results]: The Groucho corepressor is phosphorylated and downregulated in response to RTK signaling. Here we show that RTK pathways use Groucho phosphorylation as a general mechanism for inducing expression of pathway target genes encoding cell-fate determinants as well as feedback antagonists, indicating that relief of Groucho-dependent repression is an integral element of RTK signaling networks. We further demonstrate that after mitogen-activated protein kinase (MAPK) has been deactivated, sustained phosphorylation of Groucho is essential for persistent RTK-induced target gene expression and cell-fate determination in several developmental contexts. [Conclusions]: Phosphorylation of Groucho by MAPK plays a dual role in the regulation of RTK responses: (1) it mediates rapid feedback inhibition, and (2) it provides a stable memory mechanism of past MAPK activity. We propose that, in this manner, phosphorylation of Groucho enables transiently active RTK pathways to fix the spatiotemporal expression profiles of downstream targets over time. © 2011 Elsevier Ltd. All rights reserved.This research was supported by grants from the Israel Science Foundation (Center of Excellence; 180/09) and the Król Charitable Foundation to Z.P.; G.J. was supported by the Institució Catalana de Recerca i Estudis Avançats (ICREA), the Ministerio de Ciencia e Innovación (BFU2008-01875), and the Generalitat de Catalunya (2009SGR-1075); and A.O. was supported by the Israel Science Foundation. A.H. was the recipient of a PhD fellowship from the Rector of The Hebrew University of Jerusalem.Peer Reviewe

The Capicua repressor - a general sensor of RTK signaling in development and disease

Receptor tyrosine kinase (RTK) signaling pathways control multiple cellular decisions in metazoans, often by regulating the expression of downstream genes. In Drosophila melanogaster and other systems, E-twenty-six (ETS) transcription factors are considered to be the predominant nuclear effectors of RTK pathways. Here, we highlight recent progress in identifying the HMG-box protein Capicua (CIC) as a key sensor of RTK signaling in both Drosophila and mammals. Several studies have shown that CIC functions as a repressor of RTK-responsive genes, keeping them silent in the absence of signaling. Following the activation of RTK signaling, CIC repression is relieved, and this allows the expression of the targeted gene in response to local or ubiquitous activators. This regulatory switch is essential for several RTK responses in Drosophila, from the determination of cell fate to cell proliferation. Furthermore, increasing evidence supports the notion that this mechanism is conserved in mammals, where CIC has been implicated in cancer and neurodegeneration. In addition to summarizing our current knowledge on CIC, we also discuss the implications of these findings for our understanding of RTK signaling specificity in different biological processes. © 2012.This work is supported by ICREA, Ministerio de Ciencia e Innovación [grant number BFU2008-01875 to G.J.] and Generalitat de Catalunya [grant number 2009SGR-1075 to G.J.]; the National Institutes of Health and National Science Foundation to S.Y.S.; the National Institutes of Health (National Institute of General Medical Sciences) [grant number R01GM086537 to S.Y.S. and Z.P.]; the Israel Science Foundation (Centers of Excellence) [grant number 180/09 to Z.P.]; and the Król Charitable Foundation to Z.P.Peer Reviewe

Substrate-dependent control of MAPK phosphorylation in vivo

This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial.Phosphorylation of the mitogen-activated protein kinase (MAPK) is essential for its enzymatic activity and ability to control multiple substrates inside a cell. According to the current models, control of MAPK phosphorylation is independent of its substrates, which are viewed as mere sensors of MAPK activity. Contrary to this modular view of MAPK signaling, our studies in the Drosophila embryo demonstrate that substrates can regulate the level of MAPK phosphorylation in vivo. We demonstrate that a twofold change in the gene dosage of a single substrate can induce a significant change in the phosphorylation level of MAPK and in the conversion of other substrates. Our results support a model where substrates of MAPK counteract its dephosphorylation by phosphatases. Substrate-dependent control of MAPK phosphorylation is a manifestation of a more general retroactive effect that should be intrinsic to all networks with covalent modification cycles. © 2011 EMBO and Macmillan Publishers Limited.SYS acknowledges partial support by NSF via grant DMS-0718604, as well as P50 GM071508 and RO1 GM078079 grants from the NIH. GJ was supported by ICREA and by grants from MICINN (BFU2008-01875) and AGAUR (2009SGR-1075). ZP was supported by grants from the Israel Science Foundation (Centre of Excellence; 180/09) and the Król Charitable Foundation.Peer Reviewe

Data from: Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period

RNA-Seq of early follicle cells – EGFRact Rep1 Read2

Paired-end RNA-Sequencing data from early follicle cells Genotype 109-30-Gal4, UAS-mCD8::GFP, UAS-EGFR[lambda]top Replicate #1, Read

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo. 2012. © Published by The Company of Biologists Ltd.This research was supported by grants from the Israel Science Foundation (Center of Excellence; 180/09) and the Król Charitable Foundation to Z.P.; the National Institutes of Health [R01GM086537 from the National Institute of General Medical Sciences] to S.Y.S. and Z.P.; the National Science Foundation [1136913, EFRI-MIKS] to S.Y.S. and H.L.; and by ICREA, Ministerio de Ciencia e Innovación (BFU2008-01875) and Generalitat de Catalunya (2009SGR-1075) to G.J. A.H. was the recipient of a PhD Fellowship by the Rector of the Hebrew University.Peer Reviewe

Origins of Context-Dependent Gene Repression by Capicua

© 2015 Forés et al. Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.This work was funded by research grants from the Spanish Government (BFU2008-01875 and BFU2011-23611), Generalitat de Catalunya (2009SGR-1075) and Fundació La Marató de TV3 (20131730). GJ is an ICREA investigator. ZP is supported by grants from the National Institute of General Medical Sciences (NIH R01GM086537), the Israel Science Foundation (Center of Excellence; 1772/13) and the Król Charitable Foundation. ZP is an incumbent of The Lady Davis Chair in Experimental Medicine and Cancer ResearchPeer Reviewe

RNA-Seq of early follicle cells – EGFRact Rep2 Read2

Paired-end RNA-Sequencing data from early follicle cells Genotype 109-30-Gal4, UAS-mCD8::GFP, UAS-EGFR[lambda]top Replicate #2, Read