Figure 3. The long-tract distribution of cyAbrB2 on the Synechocystis genome and the repressive effect of cyAbrB2 on the gene expression.

(A) Snapshot of ChIP-seq data for cyAbrB2 and cyAbrB1 under aerobic conditions. The heatmap in the second column indicates expression fold change upon ∆cyabrb2 under aerobic conditions. Positive values (colored in red) indicate that the gene expression is higher in wildtype than in ∆cyabrb2, and negative values (colored in blue) indicate the opposite. The positions for the insertion elements are marked with red in the third column. The heatmap in the fourth column indicates GC contents. High GC contents are colored in blue and low GC contents are colored in blue. (B) GC contents and region length of cyAbrB2 binding regions (black dots). The horizontal dotted line indicates the genomic average of GC content. (C) Scatter plot of GC content and binding signal of cyAbrB2. The x-axis is the binding signal of cyAbrB2 in each 100 bp region, and the y-axis indicates GC contents within 500 bp windows sliding every 100 base pairs. CyAbrB2 binding regions are marked with red dots. (D) Histogram of genes showing the extent of occupancy (not bound, partially overlapped, or entirely overlapped) by the cyAbrB2 binding region. The gray bars indicate non-IS genes, and the count numbers of the non-IS genes are displayed on the gray bars. The black bars indicate the IS genes, and the count numbers of the IS genes are displayed above the black bars. (E) Boxplot showing fold change in gene expression by ∆cyabrb2 under aerobic conditions. Genes are classified according to the extent of occupancy by the cyAbrB2 binding region. Asterisk (*) denotes statistical significance tested by multiple comparisons of the Wilcoxon-rank test. Actual FDRs of "not bound vs 0~100%", "not bound vs 100%", and "0~100% vs 100%" are <2e-16, <2e-16, and 5e-06, respectively.

Figure 3—figure supplement 1. Overview of genome occupancy of cyAbrB2, cyAbrB1, SigE and SigA under the aerobic and microoxic conditions.

(A and B) Overview for ChIP-seq of FLAG-tagged cyAbrB2, cyAbrB1, SigE, and SigA. Y-axis indicates [normalized IP read count/normalized input read count at each 25 bp window], and x-axis indicates chromosome position. (A) Distribution of cyAbrB2, cyAbrB1, SigE, and SigA across the whole genome of Synechocystis. Aerobic (L +O₂) and dark microoxic (D – O₂) data are displayed. (B) Magnified image for chromosome position of 1550–1800 kb. ChIP-seq data of cyAbrB2, cyAbrB1, SigE, and SigA in aerobic and dark microoxic conditions are overlayed. The dots below the graph indicate the binding region of each protein calculated by peak caller.

Figure 3—figure supplement 2. Validation of procedure for ChIP-seq of FLAG-tagged cyAbrB2, SigE, and SigA.

(A) The immunoblot for inputs and immunoprecipitants (IP) of ChIP for cyAbrB2-FLAG and cyAbrB1-FLAG. Input lysate of untagged control (GT) is also loaded. Inputs equivalent to the indicated portion of IP were loaded. (B) Scatter plots showing the reproducibility of two replicates for ChIP-seq assay. ChIP-seq data of SigE, SigA, and cyAbrB2 in aerobic and microoxic conditions and ChIP-seq data of cyAbrB1 in the aerobic condition are shown. Dots indicate normalized IP read count/normalized input read count in each 100 bp window. X-axis is the value of replicate1, and y-axis is the value of replicate 2.

Figure 3—figure supplement 3. Confirmation of genomic deletion and the epitope tagging of abrB2 (#1-#3), the epitope tagging of abrB1 (#4 and #5), and deletion of sigE (#6 and #7).

Dotty lines are homologous regions between plasmids and the genome. Arrows indicate the position of check primers.

Figure 3—figure supplement 4. Relationships between GC content and binding patterns for SigE and SigA.

GC content vs ChIP enrichment score of SigA and SigE. (A) Scatter plot showing GC contents in each 100 bp vs. binding signal of SigA, SigE, and control IP. Data are displayed as in Figure 3C. (B) GC content in each 100 bp of (left) SigE peaks and non-SigE peaks, and (right) SigA peaks and non-SigA peaks.

Figure 3—figure supplement 5. cyAbrB2 and cyAbrB1 show similar binding pattern and overlapping gene regulation.

(A) Venn diagram showing overlap of the binding region of cyAbrB1 and cyAbrB2 (left), and scatter plot showing ChIP binding signal of cyAbrB2 (y-axis) and cyAbrB1(x-axis) in the aerobic condition. Data is plotted as in Figure 5A. (B) Fractions of upregulated and downregulated genes upon the ∆cyabrb2 mutant in the aerobic conditions. Fractions of all genes (left n=3608) and genes induced by cyAbrB1 knockout (right n=24) are shown. Genes induced by cyAbrB1 knockout are from the previous study (Hishida et al., 2024).