Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC7120.
Deep sequencing of HetR-bound DNA reveals novel HetR targets in Anabaena sp. strain PCC7120.
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DOI：
10.1186/s12866-014-0255-x
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发表时间：
2014-10-03
期刊：
影响因子：
4.2
通讯作者：
Golden JW
中科院分区：
文献类型：
--
作者：
Flaherty BL;Johnson DB;Golden JW
鱼腥藻(Anabaena(又称发菜))sp.菌株PCC7120，以下简称鱼腥藻，是一种蓝藻，它将大气中的氮气固定在称为异形胞的特殊细胞中。异型胞囊分化受同源二聚体转录因子HetR的调控。HetR在所有细胞中都有基础水平的表达，但在氮剥夺后早期的分化细胞中其表达增加。HetR是异形胞发育所必需的，因此是固氮和重氮化生长所必需的。HetR过表达可导致多个邻接性异囊(MCH表型)。HetR在体外与几个在异形胞中上调的基因的上游DNA片段结合，包括HetZ、hetP、HEPA、PATS、pKINE和hetR本身。HetR与其中几个基因上游的反向重复序列结合；然而，HetR与不包含该序列的启动子结合，例如PATS和pKNOE的启动子区域。我们使用染色质下拉和深度测序(CHIP-SEQ)在固定氮剥夺后6小时在体内全局识别HetR DNA靶标。我们鉴定了标记的HetR-6xHis的新的DNA结合靶标，并从这些HetR靶标序列中定义了一个一致的HetR结合位点。启动子-GFP报告融合被用来确定四个潜在的HetR靶基因的时空表达。在hetR突变株中，asr1469启动子区域在异形胞中瞬时表达，alr3758在异形囊中表达上调，alr1228在营养细胞中表达，alr2242在营养细胞中表达下调。除了识别已知的HetR靶基因hetR和hetP外，芯片序列数据还用于识别新的潜在HetR靶点和定义一致的HetR结合位点。体内对HetR调节子的CHIP-SEQ分析表明，HetR在营养细胞中除了在异形胞发育中发挥作用外，还可能发挥作用。本研究中发现的潜在HetR靶基因为进一步研究HetR在基因调控中的作用提供了新的研究对象。本文的在线版本(doi：10.1186/s12866-0140255-x)包含补充材料，授权用户可以使用。
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. 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. 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. The online version of this article (doi:10.1186/s12866-014-0255-x) contains supplementary material, which is available to authorized users.
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作者：
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DOI：
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影响因子：
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影响因子：
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通讯作者：
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