Purpose: The CCAAT-binding complex (CBC) is a heterotrimetric transcription factor conserved in all eukaryotes. The CBC specifically interacts with the pentanucleotide motif known as the CCAAT box, which is one of the most ubiquitous cis-acting elements on the eukaryotic promoters. In Aspergillus fumigatus, the CBC consists of three subunits HapB, HapC, and HapE, which are all required for specific binding to the target sequences. This regulatory has been shown to be involved in the regulation of a wide range of cellular processes, including primary and secondary metabolism, iron homeostasis, as well as azole drug resistance and virulence in mammalian hosts. Although the CCAAT motif is present in the promoter of a significant proportion of the genes in the genome of A. fumigatus, little is known about which of these are bound by the CBC. In our recent study, we investigated the genome-wide binding profile of the CBC using the chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).
Methods: A strain expressing a green fluorescent protein (GFP)-tagged HapC was generated and used for ChIP-seq analysis. Sequencing of the immunoprecipitated DNA samples was carried out with the HiSeq 2500 sequencing system, and data analysis was undertaken using a Bowtie2-MACS2 pipeline. Transcriptome profiling of a hapB knockout mutant was performed by RNA-seq, and differentially expressed genes were identified using the DESeq2 package.
Results: Genome-wide mapping of the CBC binding sites identified over 1,000 peak regions, which showed more than 5-fold enrichment compared to the input control, in the analyzed culture conditions. Bioinformatic analysis of the peak regions revealed the 5’-CSAATVR-3’ sequence as the most enriched nucleotide motif, suggesting that this motif acts as the consensus binding sequence for the CBC in vivo. Interestingly, Gene Ontrogy (GO) term enrichment analysis of the potential CBC target genes found an over-representation of gene categories related to ”transcription factor”. Consistent with this, the direct binding of the CBC was confirmed on the 5’-upstream region of approximately 50 transcription factor encoding-genes.
The impact of the CBC on the expression of its target transcription factors were investigated by comparing the transcriptome data obtained from the WT and amutant. Among the identified CBC-targets, about 40 transcription factors showed an altered expression profile in the hapB knockout mutant, indicating that the CBC both positively and negatively affects the expression of these transcription factors.
Conclusion: Our study revealed a novel insight into the genome-wide binding profile of the CBC and identified the true consensus-binding sequence for this complex. Importantly, our results suggest that the CBC acts as one of the master regulators of the transcription factors in A. fumigatus, affecting the transcription of many downstream target genes involved in highly diverse pathways
Full conference title:
- AAA 8th (2018)