de Folter Stefan, Urbanus Susan L, van Zuijlen Lisette G C, Kaufmann Kerstin, Angenent Gerco C
Business Unit Bioscience, Plant Research International, 6700 AA Wageningen, The Netherlands.
BMC Plant Biol. 2007 Sep 14;7:47. doi: 10.1186/1471-2229-7-47.
Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.
Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double Strep-tag II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP.
This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.
大多数转录因子在复合物中发挥作用，并通过与位于上游区域或内含子中的DNA基序结合来调控其靶基因。迄今为止，关于转录因子靶基因及其相应转录因子结合位点的知识仍然非常有限。两种能够在体内鉴定转录因子结合位点的相关方法是染色质免疫沉淀（ChIP）和染色质亲和纯化（ChAP）。对于ChAP，将感兴趣的蛋白质用肽或蛋白质进行标记，可用于蛋白质-DNA复合物的亲和纯化，从而鉴定靶基因。
在此，我们展示了旨在开发一种用于拟南芥MADS结构域蛋白AGAMOUS、SEPALLATA3和FRUITFULL的通用标记方法的实验结果。为此，用含有与双Strep-tag II-FLAG-tag、三重HA-tag或eGFP-tag融合的MADS-box基因的构建体转化拟南芥野生型植株，所有构建体均在组成型双35S花椰菜花叶病毒（CaMV）启动子的控制下。令人惊讶的是，在所有情况下，具有功能缺失表型的转化体数量远多于具有过表达表型的转化体。使用内源启动子而非35S CaMV可导致功能缺失表型频率的显著降低。此外，通过使用基因的天然启动子可以克服过表达策略偶尔引起的多效性缺陷。最后，展示了使用GFP抗体对携带与GFP融合的MADS-box基因基因组片段的植株进行ChAP的结果。
本研究表明，MADS-box蛋白对与小肽标签和GFP标签的融合非常敏感。此外，对于MADS-box基因嵌合版本的表达，使用与所选标签读框一致的整个基因组区域是有利的。有趣的是，尽管出乎意料，但在强35S CaMV启动子控制下使用MADS-box基因的嵌合版本似乎是获得显性负突变体的一种非常有效的方法，这是由共抑制或重组蛋白活性改变引起的。最后，我们能够通过ChAP证明AGAMOUS与其一个靶标的结合。
