Unraveling the KNOTTED1 regulatory network in maize meristems (ChIP-seq)

KNOTTED1(KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. Despite their importance for plant development, the genomic network targeted by KNOX proteins is poorly understood. Using ChIP-seq, we defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci. To understand how these binding occupancies correlate with changes in transcriptional regulation, we performed RNA-seq on immature ears and tassels, and compared expression profiles between normal and loss-of-function kn1 plants, in addition to immature leaves from normal and gain-of-function Kn1 plants. We found that 643 of the KN1 targets were modulated in one or multiple tissues, with a strong enrichment for transcription factors (including other homeobox genes) and genes participating in several hormonal pathways, most significantly auxin, implicating KN1 at the crossroads of plant hormone signaling. The loss-of-function kn1 phenotype is reminiscent of auxin mutants and kn1 mis-expression in leaves correlates with increased auxin signaling. Our results demonstrate that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function. ChIP-seq was performed using ear primordia and tassel primordia. Input DNA from each sample was used as a normalization control

Knotted1（KN1）类同源框（KNOX）转录因子在植物分生组织中发挥功能，这类由干细胞及其直接子代细胞构成的自我更新结构。尽管KNOX转录因子对植物发育至关重要，但目前学界对其靶向的基因组调控网络仍缺乏深入认知。本研究通过染色质免疫共沉淀测序（ChIP-seq），明确了玉米花序中KN1的全基因组结合位点谱（cistrome），发现KN1可结合数千个基因组位点。为探究这些结合事件与转录调控变化的关联，我们对未成熟雌穗和雄穗开展了RNA测序（RNA-seq），对比了野生型植株与功能缺失型kn1突变植株的转录表达谱；此外还对野生型与功能获得型Kn1转基因植株的未成熟叶片进行了转录组分析。结果显示，643个KN1靶基因在一种或多种组织中出现了表达调控差异，且显著富集于转录因子类基因（包含其他同源框基因）以及参与多种激素信号通路的基因，其中生长素信号通路的富集程度最高，这表明KN1位于植物激素信号转导的关键交叉节点。功能缺失型kn1突变体的表型与生长素相关突变体相似，而叶片中kn1的异常表达则会增强生长素信号通路活性。本研究结果证实，KN1在调控影响植物分生组织身份与功能的层级化基因调控网络的上游核心层级中发挥关键调控作用。本研究的ChIP-seq实验以雌穗原基和雄穗原基为实验材料，每个样本的输入基因组DNA均作为归一化对照。