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Fine Resolution Mapping of TF binding and Chromatin Interactions

ABSTRACT: Monitoring the location of transcription factors (TFs) binding to DNA is key to understanding transcriptional regulation. The main tool for mapping TF binding is ChIP-seq and its variants. However, current ChIP-based methods are hampered by at least one of the following limitations: large input requirements, low spatial resolution, and limited compatibility with high-throughput automation. Here, we describe SLIM-ChIP (Short fragment enriched, Low input, Indexed, MNase ChIP), which overcomes these challenges by combining enzymatic fragmentation of chromatin and on-bead indexing of immobilized TF-DNA complexes. We show that SLIM-ChIP reproduces high resolution binding map of yeast Reb1 similarly to the high-resolution TF mapping methods ChIP-exo and ORGANIC. Yet, SLIM-ChIP requires substantially less input material, and is fully compatible with high-throughput procedures. We further demonstrate the robustness and flexibility of SLIM-ChIP by probing Abf1 and Rap1 in yeast and CTCF in mouse embryonic stem cells. Finally, we show that the unique combination of high resolution and preservation of DNA protection patterns by SLIM-ChIP provide an additional layer of information on the chromatin landscape surrounding the bound TF. We used this information to identify a class of Reb1 sites in which the proximal -1 nucleosome tightly interacts with Reb1 and unlike in most Reb1 sites is refractory to remodeling by the RSC complex. Importantly, the interaction of Reb1 with the -1 nucleosome prevents transcription initiation and can serve as a more general mechanism for maintaining unidirectional transcription. Altogether, SLIM-ChIP is an attractive solution for mapping DNA binding proteins in a more informative context regarding their surrounding chromatin occupancy landscape at a single cell level.

ORGANISM(S): Mus musculus Saccharomyces cerevisiae

PROVIDER: GSE108948 | GEO | 2018/03/06

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.

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Fine Resolution Mapping of TF binding and Chromatin Interactions

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