Yili Yin

System-Wide Changes to SUMO Modifications in Response to Heat Shock

The small ubiquitin-like modifier protein SUMO is redistributed among many targets to mediate both short- and long-term signaling events. SUMO Status Revealed Posttranslational modification of proteins through their conjugation to small ubiquitin-like modifier (SUMO) proteins is important in the nucleus for the repair of damaged DNA and the maintenance of chromosome structure, as well as for a number of cytoplasmic processes. Although the machinery involved in attaching SUMO moieties to target proteins is well characterized, less is known about the upstream signals that trigger this modification. Golebiowski et al. designed a highly stringent, quantitative approach, involving protein purification and mass spectrometric techniques, to perform a system-wide analysis of the SUMOylation states of hundreds of proteins in HeLa cells in response to heat shock. The authors also analyzed the dynamic nature of SUMOylation in cells during the subsequent recovery phase. In addition to identifying many previously unknown substrates of SUMO-2, this proteome-wide analysis of SUMOylation revealed a rapid and dramatic redistribution of SUMO-2 among proteins involved in short- or long-term responses to heat stress. This new approach should also prove valuable in systems-wide analysis of other posttranslational modifications. Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock–induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

p53 stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products

Activation of the p53 tumour suppressor protein can lead to cell-cycle arrest or apoptosis. p53 function is controlled by the mdm2 oncogene product, which targets p53 for proteasomal degradation. In this report we demonstrate that Mdm2 induces translation of the p53 mRNA from two alternative initiation sites, giving full-length p53 and another protein with a relative molecular mass (Mr) of approximately 47K; we designate this protein as p53/47. This translation induction requires Mdm2 to interact directly with the nascent p53 polypeptide. The alternatively translated p53/47 does not contain the Mdm2-binding site and it lacks the most amino-terminal transcriptional-activation domain of p53. Increased expression of p53/47 stabilizes p53 in the presence of Mdm2, and alters the expression levels of p53-induced gene products. These results show how the interaction of Mdm2 with p53 leads to a change in the ratio of full-length p53 to p53/47 by inducing translation of both p53 proteins and the subsequent selective degradation of full-length p53. Thus, Mdm2 controls the expression levels of p53 through a dual mechanism that involves induction of synthesis and targeting for degradation.

Integration of growth and specification in chick wing digit-patterning

In the classical model of chick wing digit-patterning, the polarizing region—a group of cells at the posterior margin of the early bud—produces a morphogen gradient, now known to be based on Sonic hedgehog (Shh), that progressively specifies anteroposterior positional identities in the posterior digit-forming region. Here we add an integral growth component to this model by showing that Shh-dependent proliferation of prospective digit progenitor cells is essential for specifying the complete pattern of digits across the anteroposterior axis. Inhibiting Shh signalling in early wing buds reduced anteroposterior expansion, and posterior digits were lost because all prospective digit precursors formed anterior structures. Inhibiting proliferation also irreversibly reduced anteroposterior expansion, but instead anterior digits were lost because all prospective digit precursors formed posterior structures. When proliferation recovered in such wings, Shh transcription was maintained for longer than normal, suggesting that duration of Shh expression is controlled by a mechanism that measures proliferation. Rescue experiments confirmed that Shh-dependent proliferation controls digit number during a discrete time-window in which Shh-dependent specification normally occurs. Our findings that Shh signalling has dual functions that can be temporally uncoupled have implications for understanding congenital and evolutionary digit reductions.

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation

The chicken talpid3 mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid3 mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid3 mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid3 mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid3 mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid3 mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

ABSTRACT The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.

18-P015 3D analysis of gene expression during limb development in the Chick

involves a major collaboration between groups in Edinburgh, Dublin, Bath and London. This database will be based on EMAGE and cross-referenced to the mouse through orthologous gene pairs (http://www.emouseatlas.org/testemage/home.php). Throughout this project, the data and framework will be used to identify groups of genes that are co-expressed in important signalling regions. Conservation of these genes will be examined in the chick and mouse. This database will be made publicly available (http://www.echickatlas.org/) and will be a valuable resource to the developmental community.

The developmental mutant talpid 3 lacks primary cilia

Marc A. Willaredt, Kerstin Hasenpusch-Theil, Humphrey Gardner, Igor Kitanovic, Vera Hirschfeld-Warneken, Christian Gojak, Karin Gorgas, C. Lulu Bradford, Joachim Spatz, Stefan Woelfl, Thomas Theil, Kerry L. Tucker 1 Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany 2 Dept. of Anatomy, University of Heidelberg, Heidelberg, Germany 3 Centres for Neuroscience Research and Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom 4 Novartis Institutes forBioMedicalResearch, Cambridge, MA, United States 5 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany 6 Institute for Physical Chemistry, Dept. of Biophysical Chemistry, University of Heidelberg, Heidelberg, Germany 7 Max Planck Institute for Metals Research, Stuttgart, Germany

The developmental mutant talpid3 lacks primary cilia

Marc A. Willaredt, Kerstin Hasenpusch-Theil, Humphrey Gardner, Igor Kitanovic, Vera Hirschfeld-Warneken, Christian Gojak, Karin Gorgas, C. Lulu Bradford, Joachim Spatz, Stefan Woelfl, Thomas Theil, Kerry L. Tucker 1 Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany 2 Dept. of Anatomy, University of Heidelberg, Heidelberg, Germany 3 Centres for Neuroscience Research and Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom 4 Novartis Institutes forBioMedicalResearch, Cambridge, MA, United States 5 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany 6 Institute for Physical Chemistry, Dept. of Biophysical Chemistry, University of Heidelberg, Heidelberg, Germany 7 Max Planck Institute for Metals Research, Stuttgart, Germany