Profiling of chromatin accessibility identifies transcription factor binding sites across the genome of Aspergillus...

The identification of open chromatin regions and transcription factor binding sites (TFBs) is an important step in understanding the regulation of gene expression in diverse species. ATAC-seq is a technique used for such purpose by providing high-resolution measurements of chromatin accessibility revealed through integration of Tn5 transposase. However, the existence of cell walls in filamentous fungi and associated difficulty in purifying nuclei have precluded the routine application of this technique, leading to a lack of experimentally determined and computationally inferred data on the identity of genome-wide cis-regulatory elements (CREs) and TFBs. In this study, we constructed an ATAC-seq platform suitable for filamentous fungi and generated ATAC-seq libraries of Aspergillus niger and Aspergillus oryzae grown under a variety of conditions.

We applied the ATAC-seq assay for filamentous fungi to delineate the syntenic orthologue and differentially changed chromatin accessibility regions among different Aspergillus species, during different culture conditions, and among specific TF-deleted strains. The syntenic orthologues of accessible regions were responsible for the conservative functions across Aspergillus species, while regions differentially changed between culture conditions and TFs mutants drove differential gene expression programs. Importantly, we suggest criteria to determine TFBs through the analysis of unbalanced cleavage of distinct TF-bound DNA strands by Tn5 transposase. Based on this criterion, we constructed data libraries of the in vivo genomic footprint of A. niger under distinct conditions, and generated a database of novel transcription factor binding motifs through comparison of footprints in TF-deleted strains. Furthermore, we validated the novel TFBs in vivo through an artificial synthetic minimal promoter system.

We characterized the chromatin accessibility regions of filamentous fungi species, and identified a complete TFBs map by ATAC-seq, which provides valuable data for future analyses of transcriptional regulation in filamentous fungi.