Abstract
BackgroundAs the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints.ResultsHere we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs.ConclusionsXL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.
Highlights
Understanding of tissue-specific transcriptional regulome requires the knowledge about DNA sequencespecific chromatin interactions of transcription factors which are active in the given cell context
For a fixed source of chromatin, we chose a cell state in which numerous transcription factors (TFs) are directly interacting with chromatin in a cascade of gene regulatory actions
Since the chromatin sample is prepared from a cell population containing snapshots of these dynamic interactions, we reasoned that this would be a rich platform to assess changes in footprint depths of many TFs simultaneously
Summary
Understanding of tissue-specific transcriptional regulome requires the knowledge about DNA sequencespecific chromatin interactions of transcription factors which are active in the given cell context. Genomic TF footprinting, an alternate method for identifying occupancy of a large number of TFs in one DNase-seq or ATAC-seq sample, had the potential to circumvent many of these issues with ChIP-based assays [4, 11, 23]. Chromatin accessibility assays rely on the ability of DNA-acting enzymes to distinguish protected sites from accessible sites in the chromatin regardless of their specific DNA sequence content. These methods robustly identify cell state-specific regulatory regions which are 150 bps or larger, footprinting efforts to infer transcription factor (TF) occupancy from nucleotide-level DNase cut count (or transposase insertion count in case of ATAC-seq) profiles face different challenges. Accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints
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