Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):E3084-93. doi: 10.1073/pnas.1212457109. Epub 2012 Oct 10.
A quantitative understanding of how transcription factors interact with genomic target sites is crucial for reconstructing transcriptional networks in vivo. Here, we use Hac1, a well-characterized basic leucine zipper (bZIP) transcription factor involved in the unfolded protein response (UPR) as a model to investigate interactions between bZIP transcription factors and their target sites. During the UPR, the accumulation of unfolded proteins leads to unconventional splicing and subsequent translation of HAC1 mRNA, followed by transcription of UPR target genes. Initial candidate-based approaches identified a canonical cis-acting unfolded protein response element (UPRE-1) within target gene promoters; however, subsequent studies identified a large set of Hac1 target genes lacking this UPRE-1 and containing a different motif (UPRE-2). Using a combination of unbiased and directed microfluidic DNA binding assays, we established that Hac1 binds in two distinct modes: (i) to short (6-7 bp) UPRE-2-like motifs and (ii) to significantly longer (11-13 bp) extended UPRE-1-like motifs. Using a genetic screen, we demonstrate that a region of extended homology N-terminal to the basic DNA binding domain is required for this dual site recognition. These results establish Hac1 as the first bZIP transcription factor known to adopt more than one binding mode and unify previously conflicting and discrepant observations of Hac1 function into a cohesive model of UPR target gene activation. Our results also suggest that even structurally simple transcription factors can recognize multiple divergent target sites of very different lengths, potentially enriching their downstream target repertoire.
定量了解转录因子如何与基因组靶位点相互作用对于重建体内转录网络至关重要。在这里，我们使用 Hac1 作为模型，研究 bZIP 转录因子与其靶位点之间的相互作用。Hac1 是一种参与未折叠蛋白反应 (UPR) 的成熟碱性亮氨酸拉链 (bZIP) 转录因子，在 UPR 过程中，未折叠蛋白的积累导致 HAC1mRNA 的非常规剪接和随后翻译，随后 UPR 靶基因转录。最初基于候选的方法在靶基因启动子中鉴定出一个典型的顺式作用未折叠蛋白反应元件 (UPRE-1)；然而，随后的研究鉴定出大量缺乏这种 UPRE-1 但含有不同基序 (UPRE-2) 的 Hac1 靶基因。通过使用无偏和定向微流控 DNA 结合测定的组合，我们确定 Hac1 以两种不同的模式结合：(i) 短（6-7bp）的 UPRE-2 样基序和 (ii) 长（11-13bp）的扩展 UPRE-1 样基序。通过遗传筛选，我们证明碱性 DNA 结合结构域 N 端的扩展同源区域是这种双位点识别所必需的。这些结果确立了 Hac1 作为第一个已知采用多种结合模式的 bZIP 转录因子，并将以前相互冲突和不一致的 Hac1 功能观察结果统一到 UPR 靶基因激活的连贯模型中。我们的研究结果还表明，即使结构简单的转录因子也可以识别非常不同长度的多个发散靶位点，这可能会丰富其下游靶标谱。
