Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaenasp. strain PCC7120

Abstract

BACKGROUND: Anabaena (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. RESULTS: We 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. CONCLUSIONS: In 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.