Using Supercomputing Resources in Genomic Research

TACC resources have proven to be critical and enabling to mine cancer genomic data, genomic variants associated with human disease and polymorphic human traits, addressing biological questions otherwise non-approachable by conventional experiments. We have developed computational scripts that we use in a parallel environment to harness the capabilities of TACC HPCs, and which we have made publicly available on GitHub. In selected peer-review publications acknowledging TACC support, we have reported the association of DNA sequences able to form alternative DNA structures (or non-B DNA) with sites of chromosomal breaks leading to gross chromosomal translocations in cancer genomes, with sites of gene duplication predisposing to Parkinson’s disease, and most recently with regions of increased polymorphism in the human population. We found an exquisite correlation between the expression of selected genes and the mutational burden in cancer patients. While solving the crystal structure of a poorly characterized exonuclease, named EXO5, TACC resources enabled the assignment of a role for EXO5 in the cellular response to DNA damage, a vital pathway used by tumors to survive and grow, along with key genes whose high expression is linked to poor survival in cancer patients. Most recently, during the discovery of a nuclear role for GRB2, an adaptor protein previously thought to act only in the cytoplasm, TACC resources enabled us to test hypotheses derived from laboratory data. We were gratified to confirm the laboratory prediction that high expression of GRB2, together with its binding partner the MRE11 nuclease, carries accurate prognostic power for poor patient survival in breast cancer patients proficient in DNA homology-directed repair. These composite findings, significantly facilitated by TACC resources, have been critical to further our understanding in biological processes relevant to human disease, and to provide knowledge for the development of more precise therapeutic tools aimed at improving human health

Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation

T-cell receptor early signalling complex activation in response to interferon-α receptor stimulation

Signalling through the IFNαR (interferon-α receptor) and TCR (T-cell receptor) in Jurkat T lymphocytes results in distinct immune responses. Despite this both receptors elicit ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) phosphorylation. Vav and Slp76 are shown to be required for IFNα (interferon-α)-stimulated ERK activity. These form a subset of proteins which behave identically on stimulation of both receptors. TCR deletion abrogates IFNαR-stimulated MAPK activity, whereas the canonical JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway is unaffected. Thus recruitment of the intact TCR ESC (early signalling complex) is necessary for this downstream MAPK response. Despite using a common ESC, stimulation of the IFNαR does not produce the transcriptional response associated with TCR. Up-regulation of the MAPK pathway by IFNαR might be important to ensure that the cell responds to only one stimulant

Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen

Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor–bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain–containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2’s kinase and Shp2’s phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2–Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2

The role of SH2-B#alpha# and APS in insulin signalling

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN060761 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Indirect recruitment of the signalling adaptor Shc to the fibroblast growth factor receptor 2 (FGFR2)

International audienceThe adaptor protein Shc plays an important role in the activation of signalling pathways downstream of receptor tyrosine kinases (RTKs) regulating diverse cellular functions such as differentiation, adhesion, migration and mitogenesis. Despite being phosphorylated downstream of members of the fibroblast growth factor receptor (FGFR) family, a direct interaction of Shc with this receptor family has not been described to date. Various studies have suggested potential binding sites for the Shc PTB and/or SH2 domains on the FGFR1, but no interaction of full length Shc with these sites has been reported in vivo. We investigated the importance of the SH2 and PTB domains in recruitment of Shc to the FGFR2(IIIc) to characterise the interaction of these two proteins. Confocal microscopy revealed extensive co-localisation of Shc with the FGFR2. The PTB domain was identified as the critical component of Shc mediating membrane localisation. Data from fluorescent lifetime imaging microscopy (FLIM) revealed that the interaction of Shc and FGFR2 is indirect, suggesting that the adaptor protein forms part of a signalling complex containing the receptor. We identified the non-receptor tyrosine kinase Src as a protein potentially mediating formation of such a ternary complex. Although an interaction between Src and Shc has been described previously, we implicate the Shc SH2 domain as a novel mediator of this association. The recruitment of Shc to the FGFR2 via an indirect mechanism provides new insight into the regulation of protein assembly and activation of various signalling pathways downstream of this RTK