An Interaction Landscape of Ubiquitin Signaling.

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.

Safety evaluation of carbon tetrafluoride as an inert hyperbaric breathing gas in Sprague-Dawley rats.

Carbon tetrafluoride (CF4) is an inert gas with higher molecular weight and lower water solubility than commonly used hyperbaric breathing gases. These inert gas properties decrease time required to decompress and avoid decompression sickness after deep dives. To assess CF4 toxicity, Sprague-Dawley rats were exposed to 8 atm absolute (ATA) air (10 males, 10 females) or 8 ATA 79% CF4/21% O2 (25 males, 25 females). Exposures were 30 min daily for 5 days. Rat behavior was normal throughout the testing period. There were no gross or microscopic pathology abnormalities following repeat dose exposure. Male body weight trends were similar between groups. Female body weight trends were 0.5 ± 0.8% day-1 for hyperbaric air exposure and - 0.2 ± 0.8% day-1 for hyperbaric CF4 exposure (P = 0.01) but remained within literature cited norms. Organ weights and hematologic indices remained within or near literature normal ranges. Clinical chemistry panels showed no signs of toxicity in renal or hepatic biomarkers. Polychromatic erythrocyte micronucleus frequency showed no chromosomal damage. Comet assay showed no DNA damage in lung tissue. Females exposed to CF4 had 2.5 times greater percent tail DNA in liver tissue than controls (P = 0.009). However this result remained within the normal range of local negative controls. A bacterial reverse mutation assay with exposure to 1 ATA 79% CF4/21% O2 for 72 h was nonmutagenic in four strains of Salmonella typhimurium and one strain of Escherichia coli. Overall, there was no evidence that CF4 caused organ toxicity or genetic toxicity.

PAQMAN: Protein-nucleic acid affinity quantification by MAss spectrometry in nuclear extracts.

In recent years, various mass spectrometry-based approaches have been developed to determine global protein-DNA binding specificities using DNA affinity purifications from crude nuclear extracts. However, these assays are semi-quantitative and do not provide information about interaction affinities. We recently developed a technology that we call Protein-nucleic acid Affinity Quantification by MAss spectrometry in Nuclear extracts or PAQMAN, that can be used to determine apparent affinities between multiple nuclear proteins and a nucleic acid sequence of interest in one experiment. In PAQMAN, a series of affinity purifications with increasing bait concentrations and fixed amounts of crude nuclear extracts are combined with isobaric stable isotope labeling and quantitative mass spectrometry to generate Hill-like Kd curves for dozens of proteins in a single experiment. Here, we apply PAQMAN to determine apparent affinities for a genetic variant, rs36115365-C, which regulates TERT expression and is associated with an increased risk to develop various malignancies. Furthermore, we describe a detailed protocol for this method including important quality checks.

Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification.

In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.

An interaction proteomics survey of transcription factor binding at recurrent TERT promoter mutations.

Aberrant telomerase reactivation in differentiated cells represents a major event in oncogenic transformation. Recurrent somatic mutations in the human telomerase reverse transcriptase (TERT) promoter region, predominantly localized to two nucleotide positions, are highly prevalent in many cancer types. Both mutations create novel consensus E26 transformation-specific (ETS) motifs and are associated with increased TERT expression. Here, we perform an unbiased proteome-wide survey of transcription factor binding at TERT promoter mutations in melanoma. We observe ELF1 binding at both mutations in vitro and we show that increased recruitment of GABP is enabled by the spatial architecture of native and novel ETS motifs in the TERT promoter region. We characterize the dynamics of competitive binding between ELF1 and GABP and provide evidence for ELF1 exclusion by transcriptionally active GABP. This study thus provides an important description of proteome-wide, mutation-specific binding at the recurrent, oncogenic TERT promoter mutations.

Codon bias and gene ontology in holometabolous and hemimetabolous insects.

The relationship between preferred codon use (PCU), developmental mode, and gene ontology (GO) was investigated in a sample of nine insect species with sequenced genomes. These species were selected to represent two distinct modes of insect development, holometabolism and hemimetabolism, with an aim toward determining whether the differences in developmental timing concomitant with developmental mode would be mirrored by differences in PCU in their developmental genes. We hypothesized that the developmental genes of holometabolous insects should be under greater selective pressure for efficient translation, manifest as increased PCU, than those of hemimetabolous insects because holometabolism requires abundant protein expression over shorter time intervals than hemimetabolism, where proteins are required more uniformly in time. Preferred codon sets were defined for each species, from which the frequency of PCU for each gene was obtained. Although there were substantial differences in the genomic base composition of holometabolous and hemimetabolous insects, both groups exhibited a general preference for GC-ending codons, with the former group having higher PCU averaged across all genes. For each species, the biological process GO term for each gene was assigned that of its Drosophila homolog(s), and PCU was calculated for each GO term category. The top two GO term categories for PCU enrichment in the holometabolous insects were anatomical structure development and cell differentiation. The increased PCU in the developmental genes of holometabolous insects may reflect a general strategy to maximize the protein production of genes expressed in bursts over short time periods, e.g., heat shock proteins. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 686-698, 2015. © 2015 Wiley Periodicals, Inc.

GAT inhibition preserves cerebral blood flow and reduces oxidant damage to mitochondria in rodents exposed to extreme hyperbaric oxygen.

Oxygen breathing at elevated partial pressures (PO2's) at or more than 3 atmospheres absolute (ATA) causes a reduction in brain γ-aminobutyric acid (GABA) levels that impacts the development of central nervous system oxygen toxicity (CNS-OT). Drugs that increase brain GABA content delay the onset of CNS-OT, but it is unknown if oxidant damage is lessened because brain tissue PO2 remains elevated during hyperbaric oxygen (HBO2) exposures. Experiments were performed in rats and mice to measure brain GABA levels with or without GABA transporter inhibitors (GATs) and its influence on cerebral blood flow, oxidant damage, and aspects of mitochondrial quality control signaling (mitophagy and biogenesis). In rats pretreated with tiagabine (GAT1 inhibitor), the tachycardia, secondary rise in mean arterial blood pressure, and cerebral hyperemia were prevented during HBO2 at 5 and 6 ATA. Tiagabine and the nonselective GAT inhibitor nipecotic acid similarly extended HBO2 seizure latencies. In mice pretreated with tiagabine and exposed to HBO2 at 5 ATA, nuclear and mitochondrial DNA oxidation and astrocytosis was attenuated in the cerebellum and hippocampus. Less oxidant injury in these regions was accompanied by reduced conjugated microtubule-associated protein 1A/1B-light chain 3 (LC3-II), an index of mitophagy, and phosphorylated cAMP response element binding protein (pCREB), an initiator of mitochondrial biogenesis. We conclude that GABA prevents cerebral hyperemia and delays neuroexcitation under extreme HBO2, limiting oxidant damage in the cerebellum and hippocampus, and likely lowering mitophagy flux and initiation of pCREB-initiated mitochondrial biogenesis.

Molecular analysis of blood with micro-/nanoscale field-effect-transistor biosensors.

Rapid and accurate molecular blood analysis is essential for disease diagnosis and management. Field-effect transistor (FET) biosensors are a type of device that promise to advance blood point-of-care testing by offering desirable characteristics such as portability, high sensitivity, brief detection time, low manufacturing cost, multiplexing, and label-free detection. By controlling device parameters, desired FET biosensor performance is obtained. This review focuses on the effects of sensing environment, micro-/nanoscale device structure, operation mode, and surface functionalization on device performance and long-term stability.

Picking a nucleosome lock: Sequence- and structure-specific recognition of the nucleosome.

The nucleosome presents a formidable barrier to DNA-templated transcription by the RNA polymerase II machinery. Overcoming this transcriptional barrier in a locus-specific manner requires sequence-specific recognition of nucleosomal DNA by 'pioneer' transcription factors (TFs). Cell fate decisions, in turn, depend on the coordinated action of pioneer TFs at cell lineage-specific gene regulatory elements. Although it is already appreciated that pioneer factors play a critical role in cell differentiation, our understanding of the structural and biochemical mechanisms by which they act is still rapidly expanding. Recent research has revealed novel insight into modes of nucleosome-TF binding and uncovered kinetic principles by which nucleosomal DNA compaction affects both TF binding and residence time. Here, we review progress and argue that these structural and kinetic studies suggest new models of gene regulation by pioneer TFs.

Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma.

Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAFV600E background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.

Carbon Monoxide Levels in the Extravehicular Mobility Unit by Modeling and Operational Testing.

INTRODUCTION: Carbon monoxide (CO) is a toxic gas with potential for detriment to spaceflight operations. An analytical model was developed to investigate if a maximum CO contamination of 1 ppm in the oxygen (O2) supply reached dangerous levels during extravehicular activity (EVA). Occupational monitoring pre- and postsuited exposures provided supplementary data for review.METHODS: The analytical model estimated O2 and CO concentrations in the extravehicular mobility unit (EMU) based on O2 and CO flow rates into and out of the system. The model was based on 3 h of prebreathe at 15.2 psia, 8 h of EVA at 4.3 psia, and 1 h at 15.2 psia for suit doffing. The Coburn-Forster-Kane equation was used to calculate crewmember carboxyhemoglobin saturation (COHb%) as a function of time. Monitoring of hemoglobin CO saturation (Spco) with a CO-oximeter was conducted pre- and post-EVA during operations on the International Space Station and in ground-based analog environments.RESULTS: The model predicted a maximum PCO in the EMU of 0.061 mmHg and a maximum crewmember COHb% of 2.1%. Operational Spco measurements in mean ± SD during ground-based analog testing were 0.7% ± 1.8% pretest and 0.5% ± 1.5% posttest. Spco values on the ISS were 1.5% ± 0.7% pre-EVA and 1.1% ± 0.3% post-EVA.DISCUSSION: The model predicted that astronauts are not exposed to toxic levels of CO during EVA and operational measurements did not show significant differences between Spco levels between pre- and post-EVA.Makowski MS, Norcross JR, Alexander D, Sanders RW, Conkin J, Young M. Carbon monoxide levels in the extravehicular mobility unit by modeling and operational testing. Aerosp Med Hum Perform. 2019; 90(2):84-91.

Global profiling of protein-DNA and protein-nucleosome binding affinities using quantitative mass spectrometry.

Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein-DNA and protein-nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes.

TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A.

Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin. TRiC's binding to CSA ensures its stability and DDB1-dependent assembly into the CRLCSA complex. Consequently, loss of TRiC leads to mislocalization and depletion of CSA, as well as impaired transcription recovery following UV damage, suggesting defects in TC-NER. Furthermore, Cockayne syndrome (CS)-causing mutations in CSA lead to increased TRiC binding and a failure to compose the CRLCSA complex. Thus, we uncover CSA as a TRiC substrate and reveal that TRiC regulates CSA-dependent TC-NER and the development of CS.

Publisher correction: Functional characterization of a multi-cancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148.

This corrects the article DOI: 10.1038/ncomms15034.

SDHD Promoter Mutations Ablate GABP Transcription Factor Binding in Melanoma.

SDHD encodes subunit D of the succinate dehydrogenase complex, an integral membrane protein. Across cancer types, recurrent SDHD promoter mutations were reported to occur exclusively in melanomas, at a frequency of 4% to 5%. These mutations are predicted to disrupt consensus ETS transcription factor-binding sites and are correlated with both reduced SDHD gene expression and poor prognosis. However, the consequence of these mutations on SDHD expression in melanoma is still unclear. Here, we found that expression of SDHD in melanoma correlated with the expression of multiple ETS transcription factors, particularly in SDHD promoter wild-type samples. Consistent with the predicted loss of ETS transcription factor binding, we observed that recurrent hotspot mutations resulted in decreased luciferase activity in reporter assays. Furthermore, we demonstrated specific GABPA and GABPB1 binding to probes containing the wild-type promoter sequences, with binding disrupted by the SDHD hotspot promoter mutations in both quantitative mass spectrometry and band-shift experiments. Finally, using siRNA-mediated knockdown across multiple melanoma cell lines, we determined that loss of GABPA resulted in reduced SDHD expression at both RNA and protein levels. These data are consistent with a key role for GABPA/B1 as the critical ETS transcription factors deregulating SDHD expression in the context of highly recurrent promoter mutations in melanoma and warrant a detailed search for other recurrent promoter mutations that create or disrupt GABPA consensus sequences. Cancer Res; 77(7); 1649-61. ©2017 AACR.

A common intronic variant of PARP1 confers melanoma risk and mediates melanocyte growth via regulation of MITF.

Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ∼100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (-/GGGCCC; r2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAFV600E-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAFV600E. PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis.

Functional characterization of a multi-cancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148.

Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33. Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs. Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity. Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner. Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148. Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length. Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.

The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation.

Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.

Towards elucidating the stability, dynamics and architecture of the nucleosome remodeling and deacetylase complex by using quantitative interaction proteomics.

The nucleosome remodeling and deacetylase (NuRD) complex is an evolutionarily conserved chromatin-associated protein complex. Although the subunit composition of the mammalian complex is fairly well characterized, less is known about the stability and dynamics of these interactions. Furthermore, detailed information regarding protein-protein interaction surfaces within the complex is still largely lacking. Here, we show that the NuRD complex interacts with a number of substoichiometric zinc finger-containing proteins. Some of these interactions are salt-sensitive (ZNF512B and SALL4), whereas others (ZMYND8) are not. The stoichiometry of the core subunits is not affected by high salt concentrations, indicating that the core complex is stabilized by hydrophobic interactions. Interestingly, the RBBP4 and RBBP7 proteins are sensitive to high nonionic detergent concentrations during affinity purification. In a subunit exchange assay with stable isotope labeling by amino acids in cell culture (SILAC)-treated nuclear extracts, RBBP4 and RBBP7 were identified as dynamic core subunits of the NuRD complex, consistent with their proposed role as histone chaperones. Finally, using cross-linking MS, we have uncovered novel features of NuRD molecular architecture that complement our affinity purification-MS/MS data. Altogether, these findings extend our understanding of MBD3-NuRD structure and stability. STRUCTURED DIGITAL ABSTRACT: MBD3 physically interacts with ZNF512B, HDAC1, ZMYND8, GATAD2B, SALL4, GATAD2A, ZNF592, MTA3, ZNF687, CDK2AP1, CHD3, ZNF532, HDAC2, MTA2, CHD4, MTA1, KPNA2, CHD5, RBBP4 and RBBP7 by pull down (View interaction) CDK2AP1 physically interacts with MBD3, MTA3, HDAC2, GATAD2A, CHD4, CDK2AP1, MTA2, HDAC1, MTA1, CHD3, GATAD2B, MBD2, RBBP4 and RBBP7 by pull down (View interaction) MBD3 physically interacts with MTA2, MTA3, RBBP4, RBBP7, HDAC2, HDAC1, CHD4, CHD3 and MTA1 by cross-linking study (View interaction).

POT1 loss-of-function variants predispose to familial melanoma.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.