Fine Resolution Mapping of TF binding and Chromatin Interactions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA429230 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6453180_1.fastq.gz | Fastqsanger.gz | |||
| SRR6453180_2.fastq.gz | Fastqsanger.gz | |||
| SRR6453190_1.fastq.gz | Fastqsanger.gz | |||
| SRR6453190_2.fastq.gz | Fastqsanger.gz | |||
| SRR6453181_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal -1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcription. SLIM-ChIP is an attractive solution for mapping DNA binding proteins and charting the surrounding chromatin occupancy landscape at a single-cell level.
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Project description:Eukaryotic gene regulation relies on the binding of sequence-specific transcription factors (TFs). TFs bind chromatin transiently yet occupy their target sites by forming high-local concentration microenvironments (hubs and condensates) that increase the frequency of binding events. Despite their ubiquity, such microenvironments have been difficult to study in endogenous contexts due to technical limitations. Here, we overcome these limitations and investigate how hubs drive TF occupancy at their targets. Using a DNA binding perturbation to a hub-forming TF, Zelda, in Drosophila embryos, we find that hub properties, including the stability and frequencies of associations to targets, are key determinants of TF occupancy. Our data suggest that hub targeting is driven not just by specific DNA motif recognition, but also by a fine-tuned kinetic balance of interactions between TFs and their co-binding partners.
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