Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.
Highlights
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
The ATAC-seq assay in the filamentous fungus To identify chromatin accessibility and regulatory elements in filamentous fungi, we developed an ATAC-seq protocol for Aspergillus species by adding a step that released native nuclei by detergent lysis of protoplasts prior to the transposition step (Fig. 1a)
It can only be speculated that CreA may play an unknown activation role in gene transcriptional regulation or that the predicted binding sites contain an activated binding motif for other unknown transcription factors. These data representing the ATAC-seq landscape of Transcription factor (TF) footprints will help in the exploration of genome-wide regulation of genes active in filamentous fungi, and the data will help to discover new regulatory factors and their potential sites of action
Summary
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. 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. Transcription factors (TFs) are key regulators of biological processes that function by binding to transcriptional regulatory regions to control the expression of target genes. The ability to identify these CREs and TF motifs throughout Aspergillus genomes is an important step in understanding the regulatory functions of TF binding and gene expression. Most genome-wide TFBs in the Aspergillus genome have not been experimentally determined or computationally inferred, and the sites remain unknown
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