Genomic analysis of DNA binding and gene regulation by homologous nucleoid-associated proteins IHF and HU in Escherichia coli K12

Abstract

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ∼30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.

Genomic Looping: A Key Principle of Chromatin Organization

“…A completely new perspective on intragenic binding of H-NS was the finding that these loci often correspond to Ogasawara et al, 2010]; high-affinity binding sites determined by DNA footprint assays are indicated by a line superimposed on the ChIP-seq binding peaks. Binding sites determined by DNA footprint assays for FIS and IHF are indicated by boxes along the linear chromosome, whereas binding regions detected by ChIP-seq [Kahramanoglou et al, 2011;Prieto et al, 2012] are indicated by bars parallel to the chromosome. sites of spurious transcription (arising from weak promoters) of noncoding and antisense RNAs or due to inefficient Rho-dependent termination [Peters et al, 2012;Purtov et al, 2014;Saxena and Gowrishankar, 2011;Singh and Grainger, 2013].…”

Roles of Nucleoid-Associated Proteins in Stress-Induced Mutagenic Break Repair in Starving Escherichia coli

“…Lrp forms octomeric structures around which DNA winds, similarly to DNA wrapping around histones (de los Rios and Perona 2007). HU, IHF, Lrp, DnaA, and IciA are also global transcription regulators (Bouvier et al 1998;Oberto et al 2009;Prieto et al 2012).…”

Function of Nucleoid-Associated Proteins in Chromosome Structuring and Transcriptional Regulation

“…Wild-type HU has a significant impact on gene expression at both the transcriptional and posttranscriptional levels [Mangan et al, 2011;Oberto et al, 2009;Prieto et al, 2012], the latter reflecting its RNA binding activity [Balandina et al, 2001;; HU also possesses efficient single-stranded DNA binding activity [Kamashev et al, 2008]. Thus, HU is a highly versatile protein, acting in an architectural mode to impose structure on the nucleoid while simultaneously influencing transcription and translation.…”