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.

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.

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.

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.

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.

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.

Promoter Mutations Ablate GABP Transcription Factor Binding in Melanoma.

encodes subunit D of the succinate dehydrogenase complex, an integral membrane protein. Across cancer types, recurrent 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 gene expression and poor prognosis.

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.

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.

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.