Fine Resolution Mapping of TF binding and Chromatin Interactions. Fine Resolution Mapping of TF binding and Chromatin Interactions

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. Overall design: The dataset includes several ChIP-Seq experiments of yeast and mouse ESCs transcription factors. Reb1 (yeast) ChIP was performed in several Mnase levels and different numbers of input cells (five samples overall). Mouse CTCF ChIP was performed in several Mnase levels (five samples overall). Abf1 and Rap1 (yeast) were mapped using a mix of different Mnase levels (one sample each).

监测转录因子（transcription factors, TFs）与DNA的结合位点，是解析转录调控机制的核心所在。当前用于绘制TF结合图谱的主流技术为染色质免疫共沉淀测序（ChIP-seq）及其衍生方法。然而，现有的基于ChIP的技术方法仍存在至少一项以下局限：起始样本需求量大、空间分辨率较低，且与高通量自动化流程的兼容性有限。本研究介绍了SLIM-ChIP（Short fragment enriched, Low input, Indexed, MNase ChIP，即短片段富集型低起始量索引化微球菌核酸酶染色质免疫共沉淀测序），该技术通过结合染色质酶解片段化与固定化TF-DNA复合物的磁珠索引化策略，攻克了上述技术瓶颈。研究表明，SLIM-ChIP能够精准重现酿酒酵母Reb1蛋白的高分辨率结合图谱，其效果可媲美当前主流的高分辨率TF定位技术ChIP-exo与ORGANIC。相较于传统方法，SLIM-ChIP所需的起始样本量大幅降低，且可完全兼容高通量实验流程。本研究进一步通过对酿酒酵母Abf1、Rap1以及小鼠胚胎干细胞（embryonic stem cells, ESCs）CTCF的靶标分析，验证了SLIM-ChIP的稳定性与灵活性。最后，研究发现SLIM-ChIP兼具高分辨率与DNA保护模式保留能力的独特优势，能够为结合TF的周边染色质景观提供额外维度的信息。我们利用该信息鉴定出一类特殊的Reb1结合位点：其邻近的-1核小体与Reb1存在紧密相互作用，且与多数Reb1结合位点不同，该类位点难以被RSC复合物重塑。值得注意的是，Reb1与-1核小体的相互作用会抑制转录起始，这可能是维持单向转录的一种更为普遍的调控机制。综上，SLIM-ChIP是一种极具应用前景的DNA结合蛋白定位技术，能够在单细胞层面获取结合蛋白周边染色质占据状态的多维度信息。实验设计概要：本数据集包含酿酒酵母与小鼠胚胎干细胞（ESCs）转录因子的多组ChIP-seq实验。酿酒酵母Reb1的ChIP实验设置了不同的微球菌核酸酶（MNase）用量与起始细胞数，共包含5个样本；小鼠CTCF的ChIP实验同样设置了不同的MNase用量，共包含5个样本；酿酒酵母Abf1与Rap1的靶标定位实验则采用了不同MNase用量的混合样本，每种因子各1个样本。