Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

中文翻译：

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DFF-ChIP：一种检测和量化 RNA 聚合酶 II、转录因子和染色质之间复杂相互作用的方法


最近，我们推出了一种染色质免疫沉淀 (ChIP) 技术，利用人类 DNA 片段因子 (DFF) 在免疫沉淀之前消化 DNA (DFF-ChIP)，该技术提供转录复合物的精确位置及其与邻近核小体的相互作用。在这里，我们将该技术扩展到新的目标，并提供有关 DFF 纯化、消化条件和交联影响的有用信息。分别对 Mediator、DSIF 和 NELF 亚基进行 DFF-ChIP 分析，这些亚基不直接与 DNA 相互作用，而是与 RNA 聚合酶 II (Pol II) 相互作用。我们发现介体几乎完全与引发前复合物（PIC）相关。 DSIF 和 NELF 与参与的 Pol II 相关，此外还与 PIC 和早期起始复合物之间的潜在中间体相关。然后使用 DFF-ChIP 分析紧密结合的转录因子 CTCF 和弱得多的结合因子糖皮质激素受体 (GR) 的占据情况，无论是否存在交联。将这些结果与采用超声处理的标准 ChIP-Seq 以及利用 MNase 片段化基因组 DNA 的 CUT&RUN 的结果进行比较。我们的研究结果表明，DFF-ChIP 揭示了使用其他方法无法获得的占用细节，包括揭示相关蛋白质：蛋白质相互作用的信息。