Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea.
School of Biological Sciences and Research Institute of Basic Sciences, Seoul National University, Seoul 08826, Korea.
Genetics. 2020 Aug;215(4):1171-1189. doi: 10.1534/genetics.120.303270. Epub 2020 Jun 24.
Iron is essential for the growth of the human fungal pathogen within the vertebrate host, and iron sensing contributes to the elaboration of key virulence factors, including the formation of the polysaccharide capsule. employs sophisticated iron acquisition and utilization systems governed by the transcription factors Cir1 and HapX. However, the details of the transcriptional regulatory networks that are governed by these transcription factors and connections to virulence remain to be defined. Here, we used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome analysis (RNA-seq) to identify genes directly regulated by Cir1 and/or HapX in response to iron availability. Overall, 40 and 100 genes were directly regulated by Cir1, and 171 and 12 genes were directly regulated by HapX, under iron-limited and replete conditions, respectively. More specifically, we found that Cir1 directly controls the expression of genes required for iron acquisition and metabolism, and indirectly governs capsule formation by regulating specific protein kinases, a regulatory connection not previously revealed. HapX regulates the genes responsible for iron-dependent pathways, particularly under iron-depleted conditions. By analyzing target genes directly bound by Cir1 and HapX, we predicted the binding motifs for the transcription factors and verified that the purified proteins bind these motifs Furthermore, several direct target genes were coordinately and reciprocally regulated by Cir1 and HapX, suggesting that these transcription factors play conserved roles in the response to iron availability. In addition, biochemical analyses revealed that Cir1 and HapX are iron-containing proteins, implying that the regulatory networks of Cir1 and HapX may be influenced by the incorporation of iron into these proteins. Taken together, our identification of the genome-wide transcriptional networks provides a detailed understanding of the iron-related regulatory landscape, establishes a new connection between Cir1 and kinases that regulate capsule, and underpins genetic and biochemical analyses that reveal iron-sensing mechanisms for Cir1 and HapX in .
铁对于人源真菌病原体在脊椎动物宿主体内的生长至关重要，而铁感应有助于关键毒力因子的形成，包括多糖荚膜的形成。该病原体利用由转录因子 Cir1 和 HapX 控制的复杂铁获取和利用系统。然而，这些转录因子控制的转录调控网络的细节以及与毒力的联系仍有待确定。在这里，我们使用染色质免疫沉淀 followed by next-generation sequencing (ChIP-seq) 和转录组分析 (RNA-seq) 来鉴定直接受 Cir1 和/或 HapX 调控的基因，以响应铁的可用性。总的来说，在缺铁和铁充足的条件下，分别有 40 个和 100 个基因直接受 Cir1 调控，而分别有 171 个和 12 个基因直接受 HapX 调控。更具体地说，我们发现 Cir1 直接控制铁获取和代谢所需基因的表达，并通过调节特定的蛋白激酶间接控制荚膜的形成，这是以前未揭示的调控联系。HapX 调节与铁依赖性途径相关的基因，特别是在缺铁条件下。通过分析 Cir1 和 HapX 直接结合的靶基因，我们预测了转录因子的结合基序，并验证了纯化蛋白结合这些基序。此外，几个直接靶基因被 Cir1 和 HapX 协调地、相互地调控，这表明这些转录因子在对铁可用性的反应中发挥保守作用。此外，生化分析表明 Cir1 和 HapX 是含铁蛋白，这意味着 Cir1 和 HapX 的调控网络可能受到这些蛋白中铁的掺入的影响。总之，我们对全基因组转录网络的鉴定提供了对铁相关调控景观的详细理解，建立了 Cir1 和调节荚膜的激酶之间的新联系，并为揭示 Cir1 和 HapX 的铁感应机制提供了遗传和生化分析。
