Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis

To identify potential transient interactions between a TF and its targets, we developed an approach that can identify primary targets based either on TF-induced regulation or TF-binding, assayed in the same samples. Our studies focused on the TF bZIP1 (BASIC LEUCINE ZIPPER 1), a central integrator of cellular and metabolic signaling. To discern the mechanisms by which bZIP1 regulates a gene netowork in response to a nitrogen (N)-signal perceived in vivo, we perturbed both bZIP1 and the N-signal that it transduces in a cell-based transient expression system called TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors). To identify the genome-wide targets of bZIP1 in response to a N-signal, we transiently perturbed both the TF and the signal in the TARGET cell-based system. Specifically, bZIP1 was transiently overexpressed in root protoplasts following transfection with a 35S::GR::bZIP1 construct containing an RFP (red fluorescent protein) selectable marker gene. bZIP1 transfected cells were sequentially treated with: i) an inorganic nitrogen signal (+/-N), ii) cycloheximide (CHX) to block the synthesis of proteins, and iii) dexamethasone (DEX) to induce nuclear import of the GR::bZIP1 protein. To identify both the TF-regulated and the TF-bound genes, transcriptome analysis of transfected cells and micro-ChIP data (using anti-GR antibodies), were carried out in parallel . This submission represents transcriptome component of study.

为鉴定转录因子（TF, Transcription Factor）与其靶标间的潜在瞬时相互作用，我们开发了一种可基于转录因子诱导的调控或结合特性，在同一样本中完成检测以鉴定其初级靶标的方法。本研究聚焦于bZIP1（碱性亮氨酸拉链1，BASIC LEUCINE ZIPPER 1）——一种细胞与代谢信号传导的核心整合因子。为阐明bZIP1响应体内感知的氮（N, Nitrogen）信号以调控基因网络的分子机制，我们在名为TARGET（转录因子全基因组效应瞬时检测报告系统，Transient Assay Reporting Genome-wide Effects of Transcription factors）的基于细胞的瞬时表达系统中，对bZIP1及其转导的氮信号进行了扰动干预。为鉴定bZIP1响应氮信号的全基因组靶标，我们在该基于细胞的TARGET系统中对转录因子与信号分子同样实施了瞬时扰动。具体而言，我们将携带红色荧光蛋白（RFP, red fluorescent protein）筛选标记基因的35S::GR::bZIP1构建体转染至根原生质体中，对bZIP1进行瞬时过表达。转染bZIP1的细胞依次接受如下处理：① 无机氮信号（±N），② 环己酰亚胺（CHX, cycloheximide）以阻断蛋白质合成，③ 地塞米松（DEX, dexamethasone）以诱导GR::bZIP1蛋白的核输入。为同时鉴定转录因子调控基因与结合基因，我们并行开展了转染细胞的转录组分析与微染色质免疫沉淀（micro-ChIP）实验（使用抗GR抗体）。本提交内容即为本研究的转录组组分。