Toledo-Ortiz Gabriela, Huq Enamul, Quail Peter H
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Plant Cell. 2003 Aug;15(8):1749-70. doi: 10.1105/tpc.013839.
The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein-encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.
碱性/螺旋-环-螺旋（bHLH）蛋白是一类转录因子超家族，它们以二聚体形式结合到特定的DNA靶位点，在非植物真核生物中作为多种生物学过程的重要调控成分已得到充分表征。基于bHLH蛋白PIF3是光感受器信号网络中直接的光敏色素反应伙伴这一证据，我们对拟南芥基因组序列数据库进行了全面的计算分析，以确定bHLH家族的范围和特征。使用一组源自先前定义的共有基序的标准，我们鉴定出147个编码bHLH蛋白的基因，使其成为拟南芥中最大的转录因子家族之一。对bHLH结构域序列的系统发育分析可将这些基因分为21个亚家族。该分析所暗示的进化和潜在功能关系得到其他标准的支持，包括这些基因相对于重复基因组片段的染色体分布、可变外显子/内含子结构模式的保守性以及亚家族内预测的DNA结合活性。预计家族成员之间在DNA结合位点特异性方面存在相当大的差异，并且在保守的bHLH结构域之外的蛋白质序列中观察到明显的分歧。连同已确定的bHLH因子参与不同程度的同二聚化和异二聚化的倾向，这些观察结果表明拟南芥bHLH蛋白有可能参与广泛的组合相互作用，使其能够参与多种转录程序的调控。我们提供了来自酵母双杂交和体外结合试验的证据，表明拟南芥家族中两个相关的与光敏色素相互作用的成员PIF3和PIF4可以形成同二聚体和异二聚体，并且所有三种二聚体构型都可以特异性结合G-box DNA序列基序CACGTG。这些数据原则上与拟南芥中这种组合机制的运作一致。
