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Abstract
FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut.
Highlights
Regulation of transcription initiation by transcription factors (TFs) is a key step in controlling gene expression in all domains of life
We found that binding of nucleoidassociated proteins (NAPs) restricts FNR binding at a subset of sites, suggesting that the bacterial genome is not freely accessible for FNR binding
Our finding that less than half of the predicted FNR binding sites were occupied in vivo further challenges the utility of using bioinformatic searches alone to predict regulon structure, reinforcing the need for experimental determination of TF binding
Summary
Regulation of transcription initiation by transcription factors (TFs) is a key step in controlling gene expression in all domains of life. Genome-wide studies are revealing important features of the complexity of transcription regulation in cells not always apparent from in vitro studies. In eukaryotes, both the inhibition of TF binding by chromatin structure and the combinatorial action of multiple TFs contribute to the genome-wide pattern of TF binding and function [1,2,3,4,5]. Much less is known about how chromosome structure and combinatorial action affect bacterial TF binding and transcriptional regulation on a genome-wide scale [7]. Other studies suggest that nucleoidassociated proteins (NAPs; for example H-NS, Hu, Fis, and IHF) organize the chromosome into discrete domains and structures that may affect transcriptional regulation [7,11,12,13], but possible global effects of NAPs on TF-binding have not been systematically
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