Microsatellite instability regulates transcription factor binding and gene expression

Abstract

Microsatellites are tandemly repeated simple sequence DNA motifs widely prevalent in eukaryotic and prokaryotic genomes. In pathogenic bacteria, instability of these hypermutable loci through slipped-strand mispairing mediates the high-frequency reversible switching of phenotype expression, i.e., phase variation. Phasevariable expression of NadA, an outer membrane protein and adhesin of the pathogen Neisseria meningitidis, is mediated by changes in the number of TAAA repeats located upstream of the core promoter of nadA. Here we report that loss or gain of TAAA repeats affects the binding of the transcriptional regulatory protein IHF to the nadA promoter. Thus, phase-variable transcription of nadA potentially incorporates interplay between stochastic (mutational) and prescriptive (classical) mechanisms of gene regulation.phase variation N eisseria meningitidis is a Gram-negative encapsulated bacterium predominantly found in the human nasopharynx, where it can be a constituent of the normal microbial f lora. However, some strains are invasive and penetrate first the blood and then the meninges to cause septicemia and meningitis. N. meningitidis has evolved several environmentally induced regulatory systems to control the expression of the virulence factors that are differentially expressed during the establishment of the infection (1-3). Phase variation, which results in high-frequency switching of phenotype expression, relies on environment-independent stochastic changes occurring within repeated simple sequence DNA motifs located in coding or promoter regions of numerous genes potentially involved in pathogenicity (4). Addition or subtraction of repeated units creates frameshifts and premature stop codons (5) or alters the strength of the promoter (6 -8).The nadA gene, which encodes an outer membrane protein and adhesin (9), was recently shown to be present in 50% of the disease-associated N. meningitidis strains and in 100% of the strains belonging to hypervirulent lineages ET-5, ET-37, and cluster A4, but in only 16% of the strains isolated from healthy people (10). NadA is currently under investigation as a vaccine candidate (11). The phase-variable expression of the nadA gene was recently shown to be regulated at the transcriptional level through a variation of the number of reiterated TAAA motifs present in the repeat tract located upstream of the core promoter of the gene (12).Here we investigate the mechanism of phase variation of NadA, and in particular, the role of the TAAA microsatellite in transcriptional regulation. We show that both the TAAA repeat tract and the sequence located upstream of the microsatellite are involved in varying transcriptional activity and that the nadA promoter region also possesses multiple integration host factor (IHF)-and ferric uptake regulatory protein (Fur)-binding sites. Further, changes in the number of TAAA repeats, a requisite for phase variation, alter the binding of IHF to the nadA promoter. Thus transcription of nadA involves an interplay between mutation (slippa...

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