A Robust Analytical Pipeline for Genome-Wide Identification of the Genes Regulated by a Transcription Factor: Combinatorial Analysis Performed Using gSELEX-Seq and RNA-Seq

<div><p>For identifying the genes that are regulated by a transcription factor (TF), we have established an analytical pipeline that combines genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq and RNA-Seq. Here, SELEX was used to select DNA fragments from an <i>Aspergillus nidulans</i> genomic library that bound specifically to AmyR, a TF from <i>A</i>. <i>nidulans</i>. High-throughput sequencing data were obtained for the DNAs enriched through the selection, following which various <i>in silico</i> analyses were performed. Mapping reads to the genome revealed the binding motifs including the canonical AmyR-binding motif, CGGN₈CGG, as well as the candidate promoters controlled by AmyR. In parallel, differentially expressed genes related to AmyR were identified by using RNA-Seq analysis with samples from <i>A</i>. <i>nidulans</i> WT and <i>amyR</i> deletant. By obtaining the intersecting set of genes detected using both gSELEX-Seq and RNA-Seq, the genes directly regulated by AmyR in <i>A</i>. <i>nidulans</i> can be identified with high reliability. This analytical pipeline is a robust platform for comprehensive genome-wide identification of the genes that are regulated by a target TF.</p></div

Flowchart of the combinatorial genome-wide analysis performed using gSELEX-Seq and RNA-Seq for identifying genes regulated by TFs.

<p>Flowchart of the combinatorial genome-wide analysis performed using gSELEX-Seq and RNA-Seq for identifying genes regulated by TFs.</p

<i>A</i>. <i>nidulans</i> promoter regions selected using gSELEX-Seq.

<p><i>A</i>. <i>nidulans</i> promoter regions selected using gSELEX-Seq.</p

Flow cytometric analysis of selected DNA pools from gSELEX by using bead display.

<p>(A) Dot-plot of log fluorescence analysis. X-axis: quantified fluorescence intensity detected within the FL1 (fluorescein) channel; Y-axis: quantified fluorescence intensity detected within the FL5 (Cy5) channel. (B) Relative binding affinities measured against AmyR. The binding affinity was defined as the geometric mean of the intensity of FL1 divided by that of FL5 and the binding affinity of agdAΔ53 (a mutant of AmyR-binding DNA) against AmyR, which was set as 1.</p

Dipeptidyl peptidase IV is involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes in <i>Aspergillus aculeatus</i>

<p>We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 <i>Aspergillus aculeatus</i> T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (<i>cbhI</i>) promoter and the orotidine 5′-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and <i>cbhI</i> expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (<i>dppIV</i>) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and <i>cbhI</i> expression. The deletion of <i>dppIV</i> resulted in a significant reduction in the cellulose-responsive expression of both <i>cbhI</i> as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in <i>A. aculeatus</i>. In contrast, the <i>dppIV</i> deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in <i>A. aculeatus</i>.</p> <p>Dashed lines with arrows indicate putative signaling pathways.</p

Involvement of an SRF-MADS protein McmA in regulation of extracellular enzyme production and asexual/sexual development in <i>Aspergillus nidulans</i>

<p>SRF-MADS proteins are transcription factors conserved among eukaryotes that regulate a variety of cellular functions; however, their physiological roles are still not well understood in filamentous fungi. Effects of a mutation in <i>mcmA</i> gene that encodes the sole SRF-MADS protein in the fungus <i>Aspergillus nidulans</i> were examined by RNA sequencing. Sequencing data revealed that expression levels of cellulase genes were significantly decreased by the mutation as reported previously. However, expression levels of various hemicellulolytic enzyme genes, several extracellular protease genes, the <i>nosA</i> and <i>rosA</i> genes involved in sexual development, and AN4394 encoding an ortholog of EcdR involved in <i>Aspergillus oryzae</i> conidiation<i>,</i> were also significantly decreased by the mutation. As expected from the RNA sequencing data, the <i>mcmA</i> mutant had reduced protease production, cleistothecial development, and conidiation. This is the first report describing the involvement of SRF-MADS proteins in protease production in fungi, and asexual and sexual development in <i>Aspergillus</i>.</p> <p>McmA regulated cellulase/protease production and asexual/sexual development. The figure shows impaired development of fruiting bodies (cleistothecia) in the <i>mcmA</i> mutant.</p