For a full list of publications, check here.
DNA damage repair proteins across the tree of life
Abstract
Genome maintenance is orchestrated by a highly regulated DNA damage response with specific DNA repair pathways. Here, we investigate the phylogenetic diversity in the recognition and repair of three well-established DNA lesions, primarily repaired by base excision repair (BER) and ribonucleotide excision repair (RER): 1) 8-oxoguanine, 2) abasic site, and 3) incorporated ribonucleotide in DNA in 11 species: E. coli, B. subtilis, H. salinarum, T. brucei, T. thermophila, S. cerevisiae, S. pombe, C. elegans, H. sapiens, A. thaliana, and Z. mays. Using quantitative mass spectrometry, we identified 337 binding proteins across these species. Of these proteins, 99 were previously characterized to be involved in DNA repair. Through orthology, network, and domain analysis we linked 44 previously unconnected proteins to DNA repair. Our study presents a resource for future study of the crosstalk and evolutionary conservation of DNA damage repair across all domains of life.Systems view on the DNA damage response in Tetrahymena thermophila
Under construction
Quantitative interactomics to elucidate mechanisms of gene regulatory SNPs
Under construction
Non-coding, regulatory single nucleotide polymorphisms (SNPs) within transcription factor (TF) binding or distal DNA regulatory elements can affect local chromatin accessibility and alter the expression of gene targets through mediating different chromatin interactions. Therefore, identification of variant-specific TF interactors is of great importance to understand regulatory consequences of SNP variants. Findings on gene expression regulating SNPs, identified by massive parallel reporter assays, has opened the possibility to apply the mass spectrometry-based technique called proteome-wide analysis of SNPs (PWAS). PWAS enables identification of allele-specific DNA-TF interactions in an unbiased, sensitive manner. We have generated a large-scale PWAS dataset for 51 regulatory SNPs, performing pull downs with DNA oligos using nuclear extract of lymphoblastoids. To enable quantification of differential protein binding between variants, we applied a dimethyl labelling approach. We identify putative differential binding of proteins between variants for >85% of the tested SNPs.
Non-coding, regulatory single nucleotide polymorphisms (SNPs) within transcription factor (TF) binding or distal DNA regulatory elements can affect local chromatin accessibility and alter the expression of gene targets through mediating different chromatin interactions. Therefore, identification of variant-specific TF interactors is of great importance to understand regulatory consequences of SNP variants. Findings on gene expression regulating SNPs, identified by massive parallel reporter assays, has opened the possibility to apply the mass spectrometry-based technique called proteome-wide analysis of SNPs (PWAS). PWAS enables identification of allele-specific DNA-TF interactions in an unbiased, sensitive manner. We have generated a large-scale PWAS dataset for 51 regulatory SNPs, performing pull downs with DNA oligos using nuclear extract of lymphoblastoids. To enable quantification of differential protein binding between variants, we applied a dimethyl labelling approach. We identify putative differential binding of proteins between variants for >85% of the tested SNPs.
Profiling nucleocytoplasmic protein localization during proteotoxic stress in C. elegans
Under construction
Genetic screens in the nematode C. elegans have been used to identify cellular mechanisms that drive the toxicity of disease-related protein aggregation, and to study associated pathologies and neurotoxicity seen in human diseases. Here, we investigated whether protein translocation is a general phenomenon under proteotoxic stress by profiling the subcellular distribution of the C. elegans proteome in absence and presence of aggregation-inducing polyglutamine stretches during development. We provide an extensive resource of protein mass spectrometry data, covering larval to young adult stage, as well as cell spatial information with separation of cytosol and nuclear fractions. We show that upon induction of proteotoxic stress C. elegans undergoes global proteome remodeling, and by integrating spatial data we could distinguish distinct clusters describing changes in protein localization over time. Our approach highlights two key processes associated with proteotoxicity-induced protein relocalization: nucleocytoplasmic transport and autophagy-lysosomal pathways. This dataset provides a valuable resource for future research into these key processes, and nominates key factors that may be involved with these processes in the early stages of disease pathology.
Genetic screens in the nematode C. elegans have been used to identify cellular mechanisms that drive the toxicity of disease-related protein aggregation, and to study associated pathologies and neurotoxicity seen in human diseases. Here, we investigated whether protein translocation is a general phenomenon under proteotoxic stress by profiling the subcellular distribution of the C. elegans proteome in absence and presence of aggregation-inducing polyglutamine stretches during development. We provide an extensive resource of protein mass spectrometry data, covering larval to young adult stage, as well as cell spatial information with separation of cytosol and nuclear fractions. We show that upon induction of proteotoxic stress C. elegans undergoes global proteome remodeling, and by integrating spatial data we could distinguish distinct clusters describing changes in protein localization over time. Our approach highlights two key processes associated with proteotoxicity-induced protein relocalization: nucleocytoplasmic transport and autophagy-lysosomal pathways. This dataset provides a valuable resource for future research into these key processes, and nominates key factors that may be involved with these processes in the early stages of disease pathology.
CRISPRO
CRISPRO is a pipeline to process next generation sequencing data from
saturating mutagenesis CRISPR screens, whereby sgRNAs are mapped to
amino acids of the protein, allowing for the inference of functional
scores for amino acids in a protein. This scoring is coupled with
visualization at the primary sequence level with graphs of scores and
tracks containing domain structure and at the tertiary structure level,
where peptide fragments are aligned to existing PDB structures and
recolored in a heatmap style reflecting functional scores of amino
acids.
Click for full method.