Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli

Abstract

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these--H-NS and Fis--bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.

Figure 1.

ChIP-Seq of H-NS and Fis in E. coli K12: tracks showing binding signal (raw number of reads mapping to each basepair; signal in the form of z-scores compared with the background normal distribution; binary representation for coordinates representing binding regions) for H-NS (blue, A) and Fis (red, B). In the track showing the ‘signal’, all z-scores were subtracted by three to remove background; negative signals are not shown. The insets for H-NS show ‘long’ and ‘short’ binding regions; those for Fis show binding regions with ‘high’ and ‘low’ A/T contents.

Figure 2.

Binding of H-NS and Fis to the E. coli K12 chromosome. (A) Proportion of the genome that falls within the binding regions of H-NS (blue) and Fis (red); grey sectors of the pie charts represent the proportion of unbound regions. (B) Proportion of base pairs that are bound by H-NS only (blue), Fis only (red), or by both (green). (C) Logos representing the most significant motif associated with H-NS and Fis binding regions. (D) Proportion of binding motifs that fall within the following genomic features: operon-upstream regions (blue for H-NS, red for Fis), operon body (light blue, orange), other regions (grey). (E) Proportions of binding motifs that fall within predicted horizontally-acquired DNA (blue for H-NS, red for Fis).

Figure 3.

Length of H-NS- and Fis-binding regions. (A) Boxplot showing the distributions of the lengths of binding regions for H-NS (blue) and Fis (red) in mid-exponential phase. (B) Genomic browser (Integrated Genome Browser) view of an ∼50-kb region of the E. coli genome showing H-NS (blue) and Fis (red) binding regions in mid-exponential phase; the grey bars in the bottom panel represent annotated genes on the + (top) and the—(bottom) strands.

Figure 4.

Temporal patterns in the binding of H-NS and Fis to the chromosome. (A) Matrix showing the correlation coefficients between the binding profiles of H-NS and Fis across the four time-points studied. Note that Fis in not detectable in transition-to-stationary and stationary phases of growth. Red represents positive and green negative correlation coefficients. (B) Genomic browser (Integrated Genome Browser) view of an ∼50 kb region of the E. coli genome showing H-NS (blue) and Fis (red) binding regions across the four time-points; the grey bars in the bottom panel represent annotated genes on the + (top) and the – (bottom) strands.

Figure 5.

Protein binding and gene expression and RNA-polymerase occupancy: (A) Gene expression levels in wild-type cells of H-NS (left) and Fis (right) target- (labelled with ‘+’) and non-target (‘–’) genes. (B) RNA-polymerase occupancy in the body of target and non-target genes. (C) Gene expression fold change (log-2 scale) in the deletion strains (over wild-type) of target and non-target genes. (D) RNA-polymerase occupancy fold change in the deletion strains (over wild-type) of target and non-target genes. All plots here are for the mid-exponential phase.

Figure 6.

Length of binding regions and effect on gene expression. (A) Gene expression levels (log–2 scale) in wild-type; (B) RNA-polymerase occupancy in wild-type; (C) gene expression fold change in Δhns; (D) RNA-polymerase occupancy fold change in Δhns of genes bound by long and short H-NS binding regions. In (A–D), the distributions for genes not classified as bound by H-NS are provided as a point of reference.

Figure 7.

Fis binding features associated with differential gene expression. (A) Length of binding regions associated with Fis-bound genes which are differentially expressed (‘+’) and not (‘–’) in Δfis. (B) Number of Fis binding motifs in binding regions associated with Fis-bound genes which are differentially expressed (black) and not (grey) in Δfis. (C) Binding signal at the summit, and (D) scores assigned to the best motif within each binding region associated with Fis-bound genes which are differentially expressed (‘+’) and not (‘–’) in Δfis. (E) Distribution of the proportion of operon-upstream motifs in each binding region associated with Fis-bound genes which are differentially expressed (‘+’) and not (‘–’) in Δfis. (F) Distribution of the A/T content (50 bp on either side of the summit) of each binding region associated with Fis-bound genes which are differentially expressed (‘+’) and not (‘–’) in Δfis.

Figure 8.

Indirect effects of H-NS and Fis on gene expression. (A) Distributions of expression levels in wild-type during mid-exponential phase of genes which are down-regulated in either Δhns or Δfis or in both. Also shown are genes which are not differentially expressed in each of the two deletion mutants. (B) As above, but showing RNA-polymerase occupancy instead of gene expression levels.