A functionally conserved boundary element from the mouse HoxD locus requires GAGA factor in Drosophila

Hox genes are necessary for proper morphogenesis and organization of various body structures along the anterior-posterior body axis. These genes exist in clusters and their expression pattern follows spatial and temporal co-linearity with respect to their genomic organization. This colinearity is conserved during evolution and is thought to be constrained by the regulatory mechanisms that involve higher order chromatin structure. Earlier studies, primarily in Drosophila, have illustrated the role of chromatin-mediated regulatory processes, which include chromatin domain boundaries that separate the domains of distinct regulatory features. In the mouse HoxD complex, Evx2 and Hoxd13 are located ~9 kb apart but have clearly distinguishable temporal and spatial expression patterns. Here, we report the characterization of a chromatin domain boundary element from the Evx2-Hoxd13 region that functions in Drosophila as well as in mammalian cells. We show that the Evx2-Hoxd13 region has sequences conserved across vertebrate species including a GA repeat motif and that the Evx2-Hoxd13 boundary activity in Drosophila is dependent on GAGA factor that binds to the GA repeat motif. These results show that Hox genes are regulated by chromatin mediated mechanisms and highlight the early origin and functional conservation of such chromatin elements

FAM, a deubiquitinating enzyme essential for TGFbeta signaling controls Smad4 monoubiquitination

How growth factors can direct cell behavior depending on strength and duration of the signal remains a central unanswered question in cell and developmental biology. TGFb serves as paradigm example, as this pathway induces distinct cell fates depending on the levels of nuclear complexes formed by Smad4 with receptor phosphorylated RSmads. To serve reliably as quantitative signal, the assembly of the Smad4/RSmad complex must be not only positively regulated by receptors, but also actively kept under negative control. Yet, the factors responsible for the latter are only now starting to emerge. We found that Smad4 monoubiquitination is a central mechanism by which cells inhibit Smad4/RSmad complex assembly. By mean of siRNA screen we identified FAM (Usp9x) as a new enzyme acting as essential and evolutionary conserved component in TGFb and BMP signaling. Smad4 is monoubiquitinated in lysine 519 in vivo and this modification renders Smad4 a latent factor, impeding association with phospho- Smad2. FAM is the DUB that reverts this negative modification, re- empowering Smad4 activity. The activity of FAM is cytoplasmic and opposite to that one of Ectodermin/Tif1g(Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 K519- monoubiquitin ligase. Loss of FAM leads to increased Smad4 monoubiquitination and repression of Smad4-dependent TGF? and BMP responses, whereas loss of Ecto has opposite effects. Critically, these enzymes operate in the same pathway, being Ecto function required downstream of FAM. Intriguingly, the activity of Ecto on Smad4 is fostered by its association with P-Smad2, indicating that Ecto may also serve in vivo as “disruptase” of the Smad2/Smad4 complex. Thus, we suggest that Smad4 monoubiquitination provides a novel and powerful mechanism by which cells can quantitatively tune their responsiveness to TGFb.Come i fattori di crescita possano influenzare il comportamento cellulare sulla base dell’intensità e della durata del segnale rimane una questione irrisolta nel campo della biologia cellulare e dello sviluppo. TGFb ne è un esempio paradigmatico, in quanto questo segnale induce differenti destini cellulari in dipendenza dei livelli nucleari del complesso formato da Smad4 assieme alle Rsmads fosforilate. Per poter agire quantitativamente, la formazione dei complessi Smad4/RSmad non deve essere regolata solo positivamente dai recettori, ma anche negativamente in modo attivo. I fattori responsabili di questo controllo negativo stanno cominciando solo ora ad essere scoperti. Nel nostro studio abbiamo verificato come la monoubiquitinazione di Smad4 sia un meccanismo centrale nell’inibire la formazione del complesso Smad4/RSmad. Attraverso uno screening con siRNA abbiamo identificato FAM (Usp9x) come una nuova deubiquitinasi (DUB) che agisce come componente essenziale ed evolutivamente conservato nelle vie di segnale TGFb e BMP. Smad4 è monoubiquitinata in vivo a livello della lisina 519: questo la rende un fattore latente e ne impedisce l’associazione con fosfo-Smad2. FAM è la DUB che rimuove questa modificazione negativa ripristinando l’attività di Smad4. L’attività di FAM è citoplasmatica ed opposta a quella di Ectodermin/Tif1g(Ecto), un fattore nucleare per il quale proponiamo un ruolo come monoubiquitina ligasi di Smad4. La inattivazione di FAM porta all’aumento di monoubiquitinazione ed alla repressione delle risposte a TGFb e BMP dipendenti da Smad4; la inattivazione di Ecto ha effetti opposti. Questi enzimi agiscono nella stessa pathway, poiché la inattivazione di Ecto è dominante rispetto a quella di FAM. E’ interessante notare come l’attività di Ecto su Smad4 sia favorita dalla sua associazione con fosfo-Smad2, indicando un possibile ruolo di Ecto come “disassemblatore” del complesso Smad2/Smad4.Per questi motivi proponiamo che la monoubiquitinazione di Smad4 sia un nuovo e potente meccanismo attraverso cui le cellule possono regolare quantitativamente la loro responsività a TGFb

Integration of TGF-beta and Ras/MAPK signaling through p53 phosphorylation

During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program

Cdc42/N-WASP signaling links actin dynamics to pancreatic β cell delamination and differentiation.

Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that β cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in β cells inhibits β cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in β cells expressing constitutively active Cdc42 partially restores both delamination and β cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis

MicroRNA control of nodal signalling

MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades

FAM/USP9x, a Deubiquitinating Enzyme Essential for TGF beta Signaling, Controls Smad4 Monoubiquitination

The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation

USP15 is a deubiquitylating enzyme for receptor-activated SMADs.

The TGF\u3b2 pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGF\u3b2 and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition. Several ubiquitin ligases serve as inhibitors of R-SMADs, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGF\u3b2 and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGF\u3b2 biology