The Indispensable Role of Histone Methyltransferase Po Dot1 in Extracellular Glycoside Hydrolase Biosynthesis of Penicillium oxalicum

Abstract

Histone methylation is associated with transcription regulation, but its role for glycoside hydrolase (GH) biosynthesis is still poorly understood. We identified the histone H3 lysine 79 (H3K79)-specific methyltransferase PoDot1 in Penicillium oxalicum. PoDot1 affects conidiation by regulating the transcription of key regulators (BrlA, FlbC, and StuA) of asexual development and is required in normal hyphae septum and branch formation by regulating the transcription of five septin-encoding genes, namely, aspA, aspB, aspC, aspD, and aspE. Tandem affinity purification/mass spectrometry showed that PoDot1 has no direct interaction with transcription machinery, but it affects the expressions of extracellular GH genes extensively. The expression of genes (amy15A, amy13A, cel7A/cbh1, cel61A, chi18A, cel3A/bgl1, xyn10A, cel7B/eg1, cel5B/eg2, and cel6A/cbh2) that encode the top 10 GHs was remarkably downregulated by Podot1 deletion (ΔPodot1). Consistent with the decrease in gene transcription level, the activities of amylases and cellulases were significantly decreased in ΔPodot1 mutants in agar (solid) and fermentation (liquid) media. The repression of GH gene expressions caused by PoDot1 deletion was not mediated by key transcription factors, such as AmyR, ClrB, CreA, and XlnR, but was accompanied by defects in global demethylated H3K79 (H3K79me2) and trimethylated H3K79 (H3K79me3). The impairment of H3K79me2 on specific GH gene loci was observed due to PoDot1 deletion. The results implies that defects of H3K79 methylation is the key reason of the downregulated transcription level of GH-encoding genes and reveals the indispensable role of PoDot1 in extracellular GH biosynthesis.

Keywords: Dot1; Penicillium oxalicum; glycoside hydrolases; histone methyltransferase; regulation.

FIGURE 1

Identification of histone methyltransferase PoDot1 in P. oxalicum. (A) Phylogenetic analysis of PoDot1 orthologs. (B) The domain architecture analysis of PoDot1 orthologs. The maps were constructed with equal proportions of the respective sequences. A diagram of domains of Dot1 orthologs: DOT1 and AT_hook domains. (C,D) Subcellular localization of PoDot1. Upper left, white light; upper right, green fluorescence (green dots); bottom left, nuclear staining (blue dots); bottom right, merged. Yellow arrows indicate the location that showed the overlap of green fluorescence and nuclear staining on the merged image.

FIGURE 2

Colony morphology and conidiation of WT and PoDot1-associated mutants. (A) Morphological characteristics of the colony in 5-day-old cultures on PDA or Vogel’s agar with 2% glucose. (B) Colony diameters on Vogel’s agar with 2% glucose. (C) Levels of conidiation of the colony in 5-day-old cultures on Vogel’s agar with 2% glucose. (D) Observation of the conidiophore and mycelia of WT and PoDot1 mutants. (E) Assay of the transcription levels of brlA, stuA, and flbC. For each sample, three replicates were conducted. Gene expression copy numbers were calculated using the standard curves constructed for each gene, and the data were then normalized with the expression levels of the actin gene. Three biological triplicates were performed, and the mean values and standard deviations were calculated. Statistical analysis was performed with a one-tailed homoscedastic (equal variance) t-test, and P-values < 0.05 (^∗) were considered statistically significant.

FIGURE 3

Function enrichment of differentially expressed genes in ΔPodot1. Function enrichment of the upregulated or downregulated (more than or equal to twofold, probability ≥ 0.8) genes in ΔPodot1 compared with that of WT after the strains were cultivated in the glucose medium (A) or in the cellulose medium (B). B, C, and M indicate the GO category. B, biological process; C, cellular component; M, molecular function. Green bars, function enrichment analysis of downregulated genes. Red bars, function enrichment analysis of the upregulated genes. Blue stars indicate the same category of enrichment in both media. Blue dots indicate the clustering of three “molecular function” GO entries that are all associated with polysaccharide degradation. Significantly different expression between samples were identified through a significance test with combined thresholds (diverge probability ≥ 0.8, fold change ≥ 2) (Audic and Claverie, 1997). GO annotation and function enrichment analysis were performed by GO database (http://www.geneontology.org/) and Blast2GO with threshold at FDR ≤ 0.05 (Conesa et al., 2005).

FIGURE 4

Analysis of septa and branch formation in wild-type strains and ΔPodot1 mutants. (A) Analysis of the transcription level of five septin encoding genes. Statistical analysis was performed with a one-tailed homoscedastic (equal variance) t-test, and P-values < 0.05 (^∗) were considered statistically significant. (B) Observation of mycelial branches and interval of septa.

FIGURE 5

Comparative analysis of expression profiles of the secreted proteins in WT and ΔPodot1. (A) Clustering analysis of genes encoding the secreted proteins using Genesis (Sturn et al., 2002). The gradient color bar code at the top indicates a value at log2 fold change of expression in the treatment case to the expression in the control case. Blue dots, the top 4 extracellular glycoside hydrolases assayed in P. oxalicum secretome. (B) Expression levels of the top 10 extracellular glycoside hydrolases encoding genes. The copy number of unambiguous transcripts for each gene was normalized to FPKM.

FIGURE 6

Assay of multiple glycoside hydrolase activities of WT and PoDot1-associated mutants (ΔPodot1, RePodot1, and OEPodot1) in solid and liquid media. (A) Observation of amylolytic and cellulolytic halo around the colonies. (B) SDS-PAGE analysis of extracellular secreted proteins in liquid fermentation medium supplemented with bran and microcrystalline cellulose. (C) Amylase activities and cellulase activities in the liquid fermentation medium. Enzyme activities were normalized to the ratio of corresponding biomass (IU/mg). One enzyme activity unit was defined as the amount of enzyme required for producing 1 μmol glucose or pNP per minute under the assayed conditions. Three biological triplicates were performed for all enzyme analysis. The mean values and standard deviations were calculated. Statistical analysis was performed, and P-values < 0.05 were considered statistically significant.

FIGURE 7

Analysis transcription level of TF encoding genes and H3K79 methylation in PoDot1 mutants. (A) Expression level assay of genes encoding four TFs (clr-2/clrB, xyr1/xlnR, amyR, and cre1/creA) via qRT-PCR analysis. actin gene was used for data normalization. (B) Assay of the global histone methylation patterns at H3K79 in WT and ΔPodot1 through Western blot. Histone H3 was used as the loading control. (C) Analysis of the methylation modification level of H3K79me2 on the specific regions of four target genes (amy15A, cel7A/cbh1, cel7B/eg1, and cel61A/LPMO) through ChIP-qPCR. Equal amounts of extracted chromatin (1 mg/IP) of each sample were used for IP reactions, and 0.1 mg chromatin DNA was used as input (without IP) for each sample. The purified IP products and input DNA were subjected to quantitative PCR (qPCR). For each gene, the transcription start site (TSS) was designated as +1. Six typical regions (Region 1–Region 6) were focused: Region 1 and Region 2 were orderly located at ∼500 bp upstream (−) of the TSS. Region 3 covered the initiator and the TATA box. Region 4, Region 5, and Region 6 were orderly located in the downstream (+) of TSS, that is, in the 5′-region of the coding domain sequences (CDS), in the middle of the CDS, and in the 3′-region of the CDS, respectively. The relative enrichment of IP DNA was calculated by % of input. The values showed the means of the three biological replicates, and the error bar indicated standard deviation.

FIGURE 8

The schema of PoDot1 indirectly interacting with transcription machinery via the mediation of COMPASS. As shown by TAP results, PoDot1 has no direct interaction with transcription machinery. However, it interacts with PoSwd2, one of the subunits of COMPASS [contain subunits of Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1, Sgh1, and Set1 (Miller et al., 2001)]. It is worth noting that the subunits of RNA Pol II and general TF TFIID can be captured when PoSwd2 is used as bait to identify protein–protein interaction (Li et al., 2019). Therefore, PoDot1 indirectly interacts with transcription machinery, and this interaction was mediated by COMPASS.