Ashley J. Waardenberg

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Ashley J. Waardenberg1,6, Mirana Ramialison1,2,4,5,6, Romaric Bouveret1,2 and Richard P. Harvey1,2,3,4 1Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia 2St. Vincent's Clinical School, University of New South Wales Medicine, Kensington, New South Wales 2052, Australia 3School of Biotechnology and Biomolecular Sciences, University of New South Wales Faculty of Science, New South Wales 2052, Australia 4Stem Cells Australia, Melbourne Brain Centre, University of Melbourne, Victoria 3010, Australia Correspondence: r.harvey{at}victorchang.edu.au ↵6 These authors contributed equally to this work. ↵5 Present address: Australian Regenerative Medicine Institute, Monash University, Victoria 3800, Australia.

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

Extensive evaluation of RNA-seq methods have demonstrated that no single algorithm consistently outperforms all others. Removal of unwanted variation (RUV) has also been proposed as a method for stabilizing differential expression (DE) results. Despite this, it remains a challenge to run multiple RNA-seq algorithms to identify significant differences common to multiple algorithms, whilst also integrating and assessing the impact of RUV into all algorithms. consensusDE was developed to automate the process of identifying significant DE by combining the results from multiple algorithms with minimal user input and with the option to automatically integrate RUV. consensusDE only requires a table describing the sample groups, a directory containing BAM files or preprocessed count tables and an optional transcript database for annotation. It supports merging of technical replicates, paired analyses and outputs a compendium of plots to guide the user in subsequent analyses. Herein, we assess the ability of RUV to improve DE stability when combined with multiple algorithms and between algorithms, through application to real and simulated data. We find that, although RUV increased fold change stability between algorithms, it demonstrated improved FDR in a setting of low replication for the intersect, the effect was algorithm specific and diminished with increased replication, reinforcing increased replication for recovery of true DE genes. We finish by offering some rules and considerations for the application of RUV in a consensus-based setting. consensusDE is freely available, implemented in R and available as a Bioconductor package, under the GPL-3 license, along with a comprehensive vignette describing functionality: http://bioconductor.org/packages/consensusDE/.