Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces.
Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces.
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DOI：
10.1128/mbio.00523-16
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发表时间：
2016-04-19
期刊：
影响因子：
6.4
通讯作者：
Buttner MJ
中科院分区：
文献类型：
--
作者：
Bush MJ;Chandra G;Bibb MJ;Findlay KC;Buttner MJ
WhiB是一个蛋白质家族（WhiB样[Wbl]家族）的创始成员，该家族携带[4Fe-4S]铁硫簇，并在放线菌生物学的各个方面发挥关键作用，包括发病机制，抗生素抗性和发育控制。在链霉菌中，WhiB是发育控制的细胞分裂过程中必不可少的，导致孢子形成。Wbl蛋白的生物化学功能一直存在争议;在这里，我们开始明确地确定WhiB功能作为一个转录因子使用染色质免疫沉淀测序（ChIP-seq）在委内瑞拉链霉菌。在体内全基因组Wbl结合的第一次演示中，我们表明WhiB通过结合约240个转录单位的上游来调节孢子形成所需的关键基因的表达。引人注目的是，WhiB调节子与先前表征的WhiA调节子相同，为WhiA和WhiB突变体的相同表型提供了解释。使用ChIP-seq，我们证明了WhiA的体内DNA结合依赖于WhiB，反之亦然，表明WhiA和WhiB协同作用以控制一组共同的WhiAB靶基因的表达。最后，我们表明，突变的半胱氨酸残基协调的[4Fe-4S]簇在WhiB阻止DNA结合的WhiB和WhiA在体内。尽管WhiB样蛋白在放线菌生物学中至关重要，但其生化功能的结论性证明一直难以捉摸，并且它们一直难以研究，特别是在体外，主要是因为它们携带氧敏感的[4Fe-4S]簇。在这里，我们使用全基因组ChIP-seq来研究Wbl家族的创始成员Streptomyces WhiB的功能。这种方法的优点是，一旦蛋白质在体内与DNA交联，[4Fe-4S]簇的氧敏感性就变得无关紧要。我们的数据提供了迄今为止最令人信服的体内证据，即WhiB，并且，通过扩展，可能所有Wbl蛋白质，作为转录因子发挥作用。此外，我们表明，WhiB不独立行动，而是coregulates其调节子的孢子形成基因与合作伙伴的转录因子，WhiA。
WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo. Despite the central importance of WhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularly in vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function of Streptomyces WhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNA in vivo. Our data provide the most compelling in vivo evidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.