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. 2015 May 19;43(9):4429-46.
doi: 10.1093/nar/gkv334. Epub 2015 Apr 16.

Survey of protein-DNA interactions in Aspergillus oryzae on a genomic scale

Chao Wang  1 , Yangyong Lv  1 , Bin Wang  1 , Chao Yin  1 , Ying Lin  1 , Li Pan  2
Affiliations

Survey of protein-DNA interactions in Aspergillus oryzae on a genomic scale

Chao Wang et al. Nucleic Acids Res. .

Abstract

The genome-scale delineation of in vivo protein-DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein-DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein-DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences.

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Figures

Figure 1.
Figure 1.
Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. (A) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. (B) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level (>0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P-value < 0.05, red color). The most significant term of each group is shown by the size and color of the caption.
Figure 2.
Figure 2.
DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. (A) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). (B) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. (C) Distribution of digital footprints (FDR < 0.05 marked with blue, and 0.05 < FDR < 0.1 marked with red) relative to TSSs (black vertical line) and translation start sites (gray curved line) of genes sorted according to 5′-UTR length. (D) Expression levels (log2RPKM) for the genes observed in each of the four clusters correlated with the targeted genes. (E) The length of the 5′ UTR for the genes identified in each of the four clusters correlated with the targeted genes.
Figure 3.
Figure 3.
The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. (A) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P < 10−4, MILLIPEDE probability > 0.5). The lower panels show the average per-nucleotide DNase I cleavage patterns of plus (red line) and minus (blue line) motif sequences of the TFs and its flanking sequences. (B) The co-crystal structures of the known TFs or yeast homologues bound to the DNA recognition sites are aligned with DNase I cleavage patterns relative to the motif orientation. Upper panels: the shadows of DNA backbones and surfaces of amino acids (red) of TFs that contact with the DNA backbones, the marked depression in DNase I cleavage, are indicated in red on the crystal structure. The green color represents high-level DNase I accessibility in the crystal structure. The plus and minus motif sequences are indicated as red and blue characters, respectively. Bottom panels: the labeled amino acids in the bottom graph contact with the DNA backbones. The deoxyribose sugar rings are indicated as pentagons, and the phosphates are indicated as circles. The colors represent the same indication in upper lanes. L and R represent the binding motif sequences contacted by the left and right monomers of the TF dimer, respectively.
Figure 3.
Figure 3.
The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. (A) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P < 10−4, MILLIPEDE probability > 0.5). The lower panels show the average per-nucleotide DNase I cleavage patterns of plus (red line) and minus (blue line) motif sequences of the TFs and its flanking sequences. (B) The co-crystal structures of the known TFs or yeast homologues bound to the DNA recognition sites are aligned with DNase I cleavage patterns relative to the motif orientation. Upper panels: the shadows of DNA backbones and surfaces of amino acids (red) of TFs that contact with the DNA backbones, the marked depression in DNase I cleavage, are indicated in red on the crystal structure. The green color represents high-level DNase I accessibility in the crystal structure. The plus and minus motif sequences are indicated as red and blue characters, respectively. Bottom panels: the labeled amino acids in the bottom graph contact with the DNA backbones. The deoxyribose sugar rings are indicated as pentagons, and the phosphates are indicated as circles. The colors represent the same indication in upper lanes. L and R represent the binding motif sequences contacted by the left and right monomers of the TF dimer, respectively.
Figure 4.
Figure 4.
The DNA shape features are characteristic for the known TF motifs and its flanking sequences. (A) The plots display the average parameters of DNA shape features (MGW, propeller twist, Propeller twist, roll and helix twist) per base which are calculated in the active binding motif instances for the known TFs of the five family types. L and R represent the binding motif sequences contacted by the left and right monomers of the TF dimer, respectively. (B) Heat maps show the average MGW (upper) and Roll (down) for sequences derived from each motif instance for the known TFs of the five family types within a 10-bp flanking region. The sequences of each motif according to the average MGW were clustered and sorted into two major groups indicated with blue and red bars.
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