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High-resolution mapping of transcription factor binding sites on native chromatin

ABSTRACT: Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites.

ORGANISM(S): Saccharomyces cerevisiae Drosophila melanogaster

PROVIDER: GSE45672 | GEO | 2013/12/14

SECONDARY ACCESSION(S): PRJNA195609

REPOSITORIES: GEO

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Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.

Project description:The Nuclear Factor I (NFI) family of transcription factors (TFs) plays key roles in cellular differentiation, proliferation, and homeostasis. As such, NFI family members engage in large number of interactions with other proteins and the chromatin. However, despite their well-established significance, the NFIs interactomes, their dynamics, and their functions have not been comprehensively examined. Here, we employed complementary omics-level techniques, i.e. interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions. Our analyses included all four main NFI family members, and a less studied short isoform of NFIB (NFIB4), which lacks the DNA binding domain. We observed that, despite exhibiting some redundancy, each family member had unique high-confidence interactors and target genes, highlighting distinct roles within the transcriptional regulatory networks. The study revealed that NFIs interact with a large number of other TFs to co-regulate a broad range of regulatory networks and cellular processes. Notably, time-dependent proximity-labeling unveiled a highly dynamic nature of NFI protein-protein interaction networks and hinted at temporal modulation of NFI interactions. Furthermore, gene ontology (GO) enrichment analysis of NFI interactome and targetome revealed the involvement of NFIs in transcriptional regulation, chromatin organization, and cellular signaling pathways, and pathways related with cancer. Additionally, we observed that NFIB4 engages with proteins associated with mRNA regulation, which suggests that NFIs have roles beyond traditional DNA binding and transcriptional modulation. We propose that NFIs serve as pioneering TFs, playing critical roles in regulating other TFs and influencing a wide range of cellular processes. These insights into NFI protein-protein interactions and their dynamic, context-dependent nature provide a deeper understanding of gene regulation mechanisms and hint at the role of NFIs as master regulators.

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High-resolution mapping of transcription factor binding sites on native chromatin

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