ABSTRACT
Inflammation is characterized by a biphasic cycle consisting initially of a proinflammatory phase that is subsequently resolved by anti-inflammatory processes. Interleukin-1ß (IL-1ß) is a master regulator of proinflammation and is encoded within the same topologically associating domain (TAD) as IL-37, which is an anti-inflammatory cytokine that opposes the function of IL-1ß. Within this TAD, we identified a long noncoding RNA called AMANZI, which negatively regulates IL-1ß expression and trained immunity through the induction of IL37 transcription. We found that the activation of IL37 occurs through the formation of a dynamic long-range chromatin contact that leads to the temporal delay of anti-inflammatory responses. The common variant rs16944 present in AMANZI augments this regulatory circuit, predisposing individuals to enhanced proinflammation or immunosuppression. Our work illuminates a chromatin-mediated biphasic circuit coordinating expression of IL-1ß and IL-37, thereby regulating two functionally opposed states of inflammation from within a single TAD.
Subject(s)
Chromatin , Inflammation , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-1beta/pharmacology , Chromatin/genetics , Inflammation/genetics , Inflammation/metabolism , Cytokines , Anti-Inflammatory Agents , Interleukin-1/metabolismABSTRACT
In recent years, quantitative mass spectrometry-based interaction proteomics technology has proven very useful in identifying specific DNA-protein interactions using single pull-downs from crude lysates. Here, we applied a SILAC/TMT-based higher-order multiplexing approach to develop an interaction proteomics workflow called Protein-nucleic acid Affinity and Specificity quantification by MAss spectrometry in Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentration range of specific and control DNA baits are performed in SILAC-labeled nuclear extracts. MS1-based quantification to determine specific DNA-protein interactions is then combined with sequential TMT-based quantification of fragmented SILAC peptides, allowing the generation of Hill-like curves and determination of apparent binding affinities. We benchmarked PASMAN using the SP/KLF motif and further applied it to gain insights into two CGCG-containing consensus DNA motifs. These motifs are recognized by two BEN domain-containing proteins, BANP and BEND3, which we find to interact with these motifs with distinct affinities. Finally, we profiled the BEND3 proximal proteome, revealing the NuRD complex as the major BEND3 proximal protein complex in vivo. In summary, PASMAN represents, to our knowledge, the first higher-order multiplexing-based interaction proteomics method that can be used to decipher specific DNA-protein interactions and their apparent affinities in various biological and pathological contexts.
Subject(s)
Peptides , Proteome , Protein Binding , Proteome/analysis , Mass Spectrometry/methods , Peptides/metabolism , DNA/metabolism , Isotope Labeling/methodsABSTRACT
Transcription factor binding across the genome is regulated by DNA sequence and chromatin features. However, it is not yet possible to quantify the impact of chromatin context on transcription factor binding affinities. Here, we report a method called binding affinities to native chromatin by sequencing (BANC-seq) to determine absolute apparent binding affinities of transcription factors to native DNA across the genome. In BANC-seq, a concentration range of a tagged transcription factor is added to isolated nuclei. Concentration-dependent binding is then measured per sample to quantify apparent binding affinities across the genome. BANC-seq adds a quantitative dimension to transcription factor biology, which enables stratification of genomic targets based on transcription factor concentration and prediction of transcription factor binding sites under non-physiological conditions, such as disease-associated overexpression of (onco)genes. Notably, whereas consensus DNA binding motifs for transcription factors are important to establish high-affinity binding sites, these motifs are not always strictly required to generate nanomolar-affinity interactions in the genome.
Subject(s)
Chromatin , Transcription Factors , Chromatin/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Protein Binding , DNA/genetics , DNA/metabolism , Gene Expression Regulation , Binding Sites/genetics , Sequence Analysis, DNAABSTRACT
Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ's ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.
Subject(s)
Lipodystrophy , PPAR gamma , Humans , PPAR gamma/genetics , PPAR gamma/metabolism , Adipocytes/metabolism , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolism , Lipodystrophy/metabolism , Regulatory Sequences, Nucleic AcidABSTRACT
The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly , Chromatin/genetics , Chromatin/metabolism , CpG Islands/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Base Sequence , Cell Line, Tumor , Chromatin/chemistry , Chromatin Assembly and Disassembly/genetics , DNA Methylation , Gene Expression Regulation , Genes, Essential , Humans , Mice , Single Molecule ImagingABSTRACT
RNA-protein interactions play an important role in numerous cellular processes in health and disease. In recent years, the global RNA-bound proteome has been extensively studied, uncovering many previously unknown RNA-binding proteins. However, little is known about which particular proteins bind to which specific RNA transcript. In this review, we provide an overview of methods to identify RNA-protein interactions, with a particular focus on strategies that provide insights into the interactome of specific RNA transcripts. Finally, we discuss challenges and future directions, including the potential of CRISPR-RNA targeting systems to investigate endogenous RNA-protein interactions.
Subject(s)
Computational Biology , RNA-Binding Proteins , RNA , Clustered Regularly Interspaced Short Palindromic Repeats , Computational Biology/methods , Proteome/metabolism , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported. In this study, we comprehensively investigated the relative orientation of different subunits within the NuRD complex using multiple cross-link IP mass spectrometry (xIP-MS) experiments. Our results confirm that the core of the complex is formed by MTA, RBBP, and HDAC proteins. Assembly of a copy of MBD and GATAD2 onto this core enables binding of the peripheral CHD and CDK2AP proteins. Furthermore, our experiments reveal that not only CDK2AP1 but also CDK2AP2 interacts with the NuRD complex. This interaction requires the C terminus of CHD proteins. Our data provide a more detailed understanding of the topology of the peripheral NuRD subunits relative to the core complex. DATABASE: Proteomics data are available in the PRIDE database under the accession numbers PXD017244 and PXD017378.
Subject(s)
Cyclin-Dependent Kinases/chemistry , GATA Transcription Factors/chemistry , Histone Deacetylases/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry , Nucleosomes/ultrastructure , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Cross-Linking Reagents/chemistry , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , GATA Transcription Factors/genetics , GATA Transcription Factors/metabolism , HeLa Cells , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Mass Spectrometry/methods , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Models, Molecular , Nucleosomes/genetics , Nucleosomes/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Genetic Predisposition to Disease/genetics , Genome-Wide Association Study/methods , Melanoma/genetics , Mutation , Myxovirus Resistance Proteins/genetics , Polymorphism, Single Nucleotide , Animals , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation , Genes, Reporter/genetics , HEK293 Cells , Humans , Melanocytes/metabolism , Melanoma/pathology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Quantitative Trait Loci/genetics , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
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.
Subject(s)
High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Amino Acid Sequence , Cell Line, Tumor , Cell Nucleus/metabolism , Chromatography, Affinity/methods , DNA/chemistry , DNA/isolation & purification , DNA/metabolism , Humans , Isotope Labeling , Molecular Probe Techniques , Molecular Probes/chemistry , Molecular Probes/metabolism , Oligonucleotide Probes/chemistry , Oligonucleotide Probes/metabolism , Polymorphism, Single Nucleotide , Telomerase/analysis , Telomerase/genetics , Telomerase/isolation & purification , Telomerase/metabolismABSTRACT
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.