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.

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription

“…3d); and are targets of RpoS (the stationary phase sigma factor) identified by Weber and colleagues 38 (37 genes; P = 9.1×10 − 6 ; Fisher test compared with genes upregulated in other phases of growth). Note that difficulties in normalization of microarray data between exponential and stationary phase (as previously discussed by others 39 ) made us use data obtained from RNA-seq experiments 40 to perform the calculations on wild-type gene expression described above; though similar issues may be valid even for RNA-seq data, these should not substantially alter our qualitative conclusions. In summary, in ∆dcm, genes that seem to be already stationary phase-specific in wild-type cells are further upregulated.…”

“…RNA was extracted using Trizol reagent (Invitrogen) and mirVana miRNA isolation kit (Ambion) as reported previously 40 . Briefly, cells pelleted by centrifugation (10,000g, 10 min, 4 °C) were stored at − 80 °C until required.…”

“…The concentration was then determined using a Nanodrop ND-1000 machine (NanoDrop Technologies), and RNA quality was tested by visualization on agarose gels and by Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). For the generation of fluorescence-labelled cDNA, we used the FairPlay III Microarray Labelling Kit (Stratagene) as described previously 40 . Briefly, 1 µg of total RNA was subjected to cDNA synthesis in a reverse transcription reaction using random primers.…”

“…A list of 140 genes regulated by RpoS were obtained from Weber et al 38 . RNA-seq data for stationary and mid-exponential phase 40 were generated in our lab as reported previously and RPKM 56 values obtained from this were used.…”

“…Gene expression analyses were performed on a previously described customdesigned isothermal Agilent microarray platform, and analysed as described earlier 40 . Briefly, array data were background corrected using normexp 58 and normalization performed using VSN 59 .…”