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In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

ABSTRACT: In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis.

SUBMITTER: Grey C

PROVIDER: S-EPMC5378176 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

Grey Corinne C Clément Julie A J JA Buard Jérôme J Leblanc Benjamin B Gut Ivo I Gut Marta M Duret Laurent L de Massy Bernard B

Genome research 20170323 4

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. The ...[more]

PMID: 28336543

Similar Datasets

In vivo binding of PRDM9 reveals interaction with non-canonical genomic sites

Project description:In mouse and human meiosis, DNA double strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence specific DNA binding domain of the PRDM9 histone methyl transferase. Here we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a non-canonical recruitment of PRDM9 to sites which are devoid of both, recombination activity and the PRDM9-binding consensus motif. These sites include transcription promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset of non-canonical sites also reveals DSB-independent interactions between PRDM9 and genomic sites, which include binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot bound PRDM9 and genomic sequences located on the chromosome axis.

2017-02-08 | GSE93955 | GEO

Affinity-seq detects genome-wide PRDM9 binding sites and reveals the impact of prior chromatin modifications on mammalian recombination hotspot usage.

Project description:BackgroundGenetic recombination plays an important role in evolution, facilitating the creation of new, favorable combinations of alleles and the removal of deleterious mutations by unlinking them from surrounding sequences. In most mammals, the placement of genetic crossovers is determined by the binding of PRDM9, a highly polymorphic protein with a long zinc finger array, to its cognate binding sites. It is one of over 800 genes encoding proteins with zinc finger domains in the human genome.ResultsWe report a novel technique, Affinity-seq, that for the first time identifies both the genome-wide binding sites of DNA-binding proteins and quantitates their relative affinities. We have applied this in vitro technique to PRDM9, the zinc-finger protein that activates genetic recombination, obtaining new information on the regulation of hotspots, whose locations and activities determine the recombination landscape. We identified 31,770 binding sites in the mouse genome for the PRDM9(Dom2) variant. Comparing these results with hotspot usage in vivo, we find that less than half of potential PRDM9 binding sites are utilized in vivo. We show that hotspot usage is increased in actively transcribed genes and decreased in genomic regions containing H3K9me2/3 histone marks or bound to the nuclear lamina.ConclusionsThese results show that a major factor determining whether a binding site will become an active hotspot and what its activity will be are constraints imposed by prior chromatin modifications on the ability of PRDM9 to bind to DNA in vivo. These constraints lead to the presence of long genomic regions depleted of recombination.

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Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response.

Project description:Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κΒ and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κΒ that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.

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Mapping ERβ genomic binding sites reveals unique genomic features and identifies EBF1 as an ERβ interactor.

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Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

Project description:Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.

| S-EPMC4466390 | biostudies-literature

A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites.

Project description:Defining the hardwiring of transcription factors to their cognate genomic binding sites is essential for our understanding of biological processes. We used scanning chromatin immunoprecipitation to identify in vivo binding regions (E boxes) for c-Myc in three target genes as a model system. Along with other c-Myc target genes that have been validated by chromatin immunoprecipitation, we used the publicly available genomic sequences to determine whether experimentally derived in vivo binding sites might be predictable from nonexonic sequence conservation across species. Our studies revealed two classes of target genomic binding sites. Although the majority of target genes studied [class I: B23 (NPM1), CAD, CDK4, cyclin D2, ID2, LDH-A, MNT, PTMa, ODC, NM23B, nucleolin, prohibitin, SHMT1, and SHMT2] demonstrate significant sequence conservation of the E boxes and flanking regions, several genes (cyclin B1, JPO1, and PRDX3) belong to a second class (class II) that does not display sequence conservation at and around the site of c-Myc binding. On the basis of our model, we propose a strategy for predicting transcription factor binding sites using phylogenetic sequence comparisons, which will select potential class I target genes among the many emerging candidates from DNA-microarray studies for experimental validation by chromatin immunoprecipitation.

| S-EPMC154342 | biostudies-literature

Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters.

Project description:Hypoxia-inducible factor (HIF) directs an extensive transcriptional cascade that transduces numerous adaptive responses to hypoxia. Pan-genomic analyses, using chromatin immunoprecipitation and transcript profiling, have revealed large numbers of HIF-binding sites that are generally associated with hypoxia-inducible transcripts, even over long chromosomal distances. However, these studies do not define the specific targets of HIF-binding sites and do not reveal how induction of HIF affects chromatin conformation over distantly connected functional elements. To address these questions, we deployed a recently developed chromosome conformation assay that enables simultaneous high-resolution analyses from multiple viewpoints. These assays defined specific long-range interactions between intergenic HIF-binding regions and one or more promoters of hypoxia-inducible genes, revealing the existence of multiple enhancer-promoter, promoter-enhancer, and enhancer-enhancer interactions. However, neither short-term activation of HIF by hypoxia, nor long-term stabilization of HIF in von Hippel-Lindau (VHL)-defective cells greatly alters these interactions, indicating that at least under these conditions, HIF can operate on preexisting patterns of chromatin-chromatin interactions that define potential transcriptional targets and permit rapid gene activation by hypoxic stress.

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Mechanisms of noncanonical binding dynamics in multivalent protein-protein interactions.

Project description:Protein multivalency can provide increased affinity and specificity relative to monovalent counterparts, but these emergent biochemical properties and their mechanistic underpinnings are difficult to predict as a function of the biophysical properties of the multivalent binding partners. Here, we present a mathematical model that accurately simulates binding kinetics and equilibria of multivalent protein-protein interactions as a function of the kinetics of monomer-monomer binding, the structure and topology of the multidomain interacting partners, and the valency of each partner. These properties are all experimentally or computationally estimated a priori, including approximating topology with a worm-like chain model applicable to a variety of structurally disparate systems, thus making the model predictive without parameter fitting. We conceptualize multivalent binding as a protein-protein interaction network: ligand and receptor valencies determine the number of interacting species in the network, with monomer kinetics and structural properties dictating the dynamics of each species. As predicted by the model and validated by surface plasmon resonance experiments, multivalent interactions can generate several noncanonical macroscopic binding dynamics, including a transient burst of high-energy configurations during association, biphasic equilibria resulting from interligand competition at high concentrations, and multiexponential dissociation arising from differential lifetimes of distinct network species. The transient burst was only uncovered when extending our analysis to trivalent interactions due to the significantly larger network, and we were able to predictably tune burst magnitude by altering linker rigidity. This study elucidates mechanisms of multivalent binding and establishes a framework for model-guided analysis and engineering of such interactions.

| S-EPMC6926015 | biostudies-literature

In vivo genomic binding sites for the Drosophila CTCF Insulator

Project description:Chromatin immunopurification with anti-CTCF was used to identify in vivo binding sites for CTCF in approximately 3% of the Drosophila genome using amplicon-based tiling arrays. This includes the Bithorax-complex, a known site of CTCF activity, the Antp-Complex, the Achaete scute-Complex and the Adh region. Reproducible binding identified new CTCF sites in the Adh region and the Bx-C. Keywords: ChIP-chip

2007-06-01 | GSE7351 | GEO

A massively parallel reporter assay reveals context-dependent activity of homeodomain binding sites in vivo.

Project description:Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by 2 or 3 nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we used a massively parallel reporter assay to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assayed inactivating mutations in more than 1700 TF binding sites and found that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict 3-bp spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites showed that, on average, changes in TF binding site affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple cis-regulatory elements (CREs). Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types.

| S-EPMC6169884 | biostudies-literature

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