Benhamed Moussa, Martin-Magniette Marie-Laure, Taconnat Ludivine, Bitton Frédérique, Servet Caroline, De Clercq Rebecca, De Meyer Björn, Buysschaert Caroline, Rombauts Stéphane, Villarroel Raimundo, Aubourg Sébastien, Beynon Jim, Bhalerao Rishikesh P, Coupland George, Gruissem Wilhelm, Menke Frank L H, Weisshaar Bernd, Renou Jean-Pierre, Zhou Dao-Xiu, Hilson Pierre
Institut de Biotechnologie des Plantes, UMR 8618, Centre National de la Recherche Scientifique, Université de Paris Sud 11, 91405 Orsay, France.
Plant J. 2008 Nov;56(3):493-504. doi: 10.1111/j.1365-313X.2008.03606.x. Epub 2008 Sep 18.
We have assembled approximately 20 000 Arabidopsis thaliana promoter regions, compatible with functional studies that require cloning and with microarray applications. The promoter fragments can be captured as modular entry clones (MultiSite Gateway format) via site-specific recombinational cloning, and transferred into vectors of choice to investigate transcriptional networks. The fragments can also be amplified by PCR and printed on glass arrays. In combination with immunoprecipitation of protein-DNA complexes (ChIP-chip), these arrays enable characterization of binding sites for chromatin-associated proteins or the extent of chromatin modifications at genome scale. The Arabidopsis histone acetyltransferase GCN5 associated with 40% of the tested promoters. At most sites, binding did not depend on the integrity of the GCN5 bromodomain. However, the presence of the bromodomain was necessary for binding to 11% of the promoter regions, and correlated with acetylation of lysine 14 of histone H3 in these promoters. Combined analysis of ChIP-chip and transcriptomic data indicated that binding of GCN5 does not strictly correlate with gene activation. GCN5 has previously been shown to be required for light-regulated gene expression and growth, and we found that GCN5 targets were enriched in early light-responsive genes. Thus, in addition to its transcriptional activation function, GCN5 may play an important role in priming activation of inducible genes under non-induced conditions.
我们已经收集了大约20000个拟南芥启动子区域，适用于需要克隆的功能研究和微阵列应用。启动子片段可通过位点特异性重组克隆捕获为模块化入门克隆（MultiSite Gateway格式），并转移到所选载体中以研究转录网络。这些片段也可以通过PCR扩增并打印在玻璃阵列上。与蛋白质-DNA复合物的免疫沉淀（ChIP-chip）相结合，这些阵列能够在基因组规模上表征染色质相关蛋白的结合位点或染色质修饰的程度。拟南芥组蛋白乙酰转移酶GCN5与40%的测试启动子相关。在大多数位点，结合不依赖于GCN5溴结构域的完整性。然而，溴结构域的存在对于与11%的启动子区域结合是必要的，并且与这些启动子中组蛋白H3赖氨酸14的乙酰化相关。ChIP-chip和转录组数据的联合分析表明，GCN5的结合与基因激活并不严格相关。先前已证明GCN5是光调节基因表达和生长所必需的，我们发现GCN5的靶标在早期光响应基因中富集。因此，除了其转录激活功能外，GCN5可能在非诱导条件下引发可诱导基因的激活中发挥重要作用。
