Tankyrase 1 influences telomere recombination, stability of the NHEJ protein DNA-PKcs and genomic integrity

2011 Spring.Includes bibliographical references.The Poly(ADP-ribosyl)ating Polymerase (PARP) family of enzymes has gained considerable attention recently due to the success of inhibiting their activities in breast cancers with BRCA 1/2 deficient backgrounds. PARPs serve as key regulators of protein recruitment, stability and activity in specific intracellular pathways including DNA-repair, telomere stability, transcription factor regulation and mitotic integrity. The PARP family member, PARP-5a, otherwise known as tankyrase 1 is unique in that it lacks a DNA-binding domain and interacts with proteins specifically. First found to regulate telomere length by promoting access to telomerase, tankyrase 1 has since become associated with a multitude of critical cellular processes. In our studies investigating the role of DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs) and tankyrase 1 at telomeres, we find that tankyrase 1 is required for the suppression of sister chromatid recombination events at the telomere and that the leucine zipper domain of DNA-PKcs is necessary for accurate end-capping function. Interestingly, during our investigation we also identified a link between the stability of the DNA-PKcs protein and tankyrase 1. We find that under conditions in which tankyrase 1 is depleted or catalytically inhibited, DNA-PKcs becomes a substrate for proteasome mediated degradation. The depletion of tankyrase 1 by siRNA-mediated knockdown or PARP inhibition resulted in the failure of DNA-PKcs function in both telomere end-capping and the DNA damage response following exposure to ionizing radiation; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberrations and telomere fusions. Further, we find that the loss of DNA-PKcs is not coupled with depletion of Ku70, Ku80 or the PI3-kinase ATM, illustrating that tankyrase 1 acts to regulate DNA-PKcs specifically. Taken together, we identify important and novel roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

<p>Abstract</p> <p>Background</p> <p>Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.</p> <p>Results</p> <p>We show here that SYF cells (mouse fibroblasts lacking the NTKs Src, Yes and Fyn) exhibit a low level of basal tyrosine phosphorylation at focal adhesions. FIT-mediated tyrosine phosphorylation of NTK substrates p130Cas, paxillin and FAK and cortactin was observed at focal adhesions, while FIT-mediated phosphorylation of cortactin was also seen at the cell periphery. Phosphorylation of p130Cas in SYF cells led to activation of Rac1 and increased membrane ruffling and lamellipodium formation, events associated with cell migration. We also found that the kinase activity of Src and not FAK is essential for phosphorylation of p130Cas when the three proteins exist as a complex in focal adhesions.</p> <p>Conclusion</p> <p>These results demonstrate that tyrosine phosphorylation of p130Cas is sufficient for its localization to focal adhesions and for activation of downstream signaling events associated with cell migration. FIT provides a valuable tool to evaluate the contribution of individual components of the response to signals with multiple outputs, such as activation of NTKs.</p

Configuring robust DNA strand displacement reactions for in situ molecular analyses

The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells

Détermination du mécanisme de discrimination entre l'ADN spécifique et non-spécifique par les facteurs de transcription B/HLH/LZ

Le réseau de facteurs de transcription c-Myc/Max/Mad contrôle la transcription de plus de 1500 gènes à l'intérieur du génome humain. Dans ce réseau, les domaines basic-Helix-Loop-Helix-Leucine Zipper (b/HLH/LZ) sont responsables de la reconnaissance moléculaire entre les protéines et de la liaison au promoteur des gènes cibles. Par contre, les détails du mécanisme de liaison spécifique à l'ADN demeurent inconnus. Dans cette thèse, nous révélons le mécanisme de discrimination entre les séquences spécifiques et non-spécifiques par les facteurs de transcription b/HLH/LZ. Article 1: À partir de la détermination par Résonance Magnétique Nucléaire (RMN) de la structure et la dynamique du dimère b/HLH/LZ de Max en solution et en absence d'ADN, nous avons proposé un mécanisme pour la formation spécifique et réversible de complexes protéine/ADN par cette famille de facteurs de transcription. En solution et en absence d'ADN, les domaines H1LH2/LZ sont bien repliés alors que la région basique est majoritaire non-repliée. Nous avons démontré que H1 déploie des mouvements de grande amplitude dans l'échelle de temps de la nanoseconde. Nous avons proposé que ces mouvements résultent d'un "cluster" de chaînes-latérales basiques à l'intérieur des conformères partiellement repliés. Via cette structure, nous avons proposé un mécanisme de liaison à l'ADN composé d'un mélange de sélection conformationelle et de repliement assisté par l'ADN. Premièrement, l'état partiellement replié lie directement le sillon majeur de l'ADN par mode de sélection conformationelle. Cette étape permet la liaison de séquences d'ADN spécifique (ASp) et non-spécifique (ANSp). Ensuite, la région basique subit un repliement assisté par l'ADN de sa structure secondaire en hélice alpha. Nous avons proposé que le repliement et la stabilisation de cette région basique soient responsables de la discrimination entre les ASp et ANSp. Lorsque le complexe-ANSp est formé, la région basique est majoritairement non-repliée à cause des interactions non-favorables entre les chaînes-latérales de la protéine et l'ADN ce qui donne lieu à des complexes de faible affinité avec une haute probabilité de se dissocier. Dans le cas d'un complexe-ASp, les interactions favorables entre les chaînes-latérales clés de la région basique (e.g. E12) et l'ADN favorise l'équilibre vers la forme optimalement replié de la région basique et du complexe de haute affinité. Article 2: Afin de vérifier notre mécanisme proposé, nous avons réalisé des études de structure, stabilité thermodynamique et de dynamique de la protéine Max en complexe avec ASp (5'-CACGTG-3') et ANSp (5'-GGATCC-3') par Dichroïsme Circulaire (CD) et RMN. Nous démontrons qu'il y a une différence apparente d'affinité entre l'ASp (KD=110 8) et l'ANSp (KD=1106) pour Max. Aussi, le complexe-ASp contient plus de structure secondaire par rapport au complexe-ANSp. Ces résultats supportent notre mécanisme où la région basique du complexe-ANSp est moins repliée. Par RMN, nous démontrons que la région basique du complexe-ANSp est plus dynamique et flexible que le complexe-ASp. Une analyse moléculaire de l'interface de liaison protéine/ADN montre que l'interaction clé entre la Glutamate E12 et les nucléotides CA de l'ASp est absente dans le cas de l'ANSp. Ceci mène à la dénaturation de la région basique dans le complexe NSD et permet la dissociation de l'ADN. Dans une vue d'ensemble, cette thèse présente un mécanisme pour la discrimination des Asp des ANSp par les facteurs de transcription b/HLH/LZ. Ces informations sont importantes pour le design rationnel de nouveaux agents thérapeutiques afin de combattre les cancers impliquant l'oncoprotéine c-Myc

GASZ Is Essential for Male Meiosis and Suppression of Retrotransposon Expression in the Male Germline

Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage