Abstract
BackgroundAnabaena (also Nostoc) sp. strain PCC7120, hereafter Anabaena, is a cyanobacterium that fixes atmospheric N2 in specialized cells called heterocysts. Heterocyst differentiation is regulated by a homodimeric transcription factor, HetR. HetR is expressed at a basal level in all cells but its expression increases in differentiating cells early after nitrogen deprivation. HetR is required for heterocyst development, and therefore nitrogen fixation and diazotrophic growth. Overexpression of HetR leads to multiple contiguous heterocysts (Mch phenotype). HetR binds in vitro to DNA fragments upstream of several genes upregulated in heterocysts, including hetZ, hetP, hepA, patS, pknE, and hetR itself. HetR binds an inverted repeat sequence upstream of a few of these genes; however, HetR binds to promoters that do not contain this sequence, such as the promoter regions for patS and pknE.ResultsWe employed chromatin pull-down and deep sequencing (ChIP-seq) to globally identify HetR DNA targets in vivo at six hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Promoter-gfp reporter fusions were used to determine the spatiotemporal expression of four potential HetR-target genes. The promoter region for asr1469 was expressed transiently in differentiating heterocysts, alr3758 was upregulated in heterocysts, asl2028 was expressed in vegetative cells, and alr2242 was derepressed in vegetative cells of a hetR mutant strain.ConclusionsIn addition to identifying known HetR target genes hetR and hetP, the ChIP-seq data were used to identify new potential HetR targets and to define a consensus HetR-binding site. The in vivo ChIP-seq analysis of HetR’s regulon suggests a possible role for HetR in vegetative cells in addition to its role in heterocyst development. The potential HetR target genes identified in this study provide new subjects for future work on the role of HetR in gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0255-x) contains supplementary material, which is available to authorized users.
Highlights
Anabaena ( Nostoc) sp. strain PCC7120, hereafter Anabaena, is a cyanobacterium that fixes atmospheric N2 in specialized cells called heterocysts
We employed a combination of Chromatin immunoprecipitation or pull-down (ChIP)-seq and RNA-seq to examine the HetR regulon at six hours after nitrogen deprivation, when HetR is known to be involved in the activation of some heterocyst-specific genes
Future ChIP-seq studies from different times during heterocyst development may result in identifying additional HetR targets because it is likely that HetR binding to DNA is affected by HetR concentration and influenced by other factors that may be present at different times or under different conditions than those used in this study
Summary
Anabaena ( Nostoc) sp. strain PCC7120, hereafter Anabaena, is a cyanobacterium that fixes atmospheric N2 in specialized cells called heterocysts. Strain PCC7120, hereafter Anabaena, is a cyanobacterium that fixes atmospheric N2 in specialized cells called heterocysts. Anabaena and Nostoc species fix atmospheric N2 into ammonia in specialized nitrogen-fixing cells called heterocysts at spaced intervals along filaments of photosynthetic vegetative cells. Strain PCC7120, overexpression of HetR results in multiple contiguous heterocysts (Mch phenotype) in. HetR binds in vitro to DNA fragments upstream of several genes upregulated in heterocysts, including hetZ, hetP, hepA, pknE, patS, and hetR itself [8,9,10,11]. A study that identified the HetR binding site upstream of hetZ suggested that the HetR binding site is related to the imperfect palindrome GGGTCTAgCCCagCA [10], but this site is not upstream of all known HetR targets, including the patS gene, which is involved in heterocyst pattern regulation [12,13,14]
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