Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae (HTS)

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified S. cerevisiae Fkh1 and Fkh2 as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homo-dimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.叉头框（Forkhead Box, Fox）蛋白共享叉头结构域——一种翼螺旋型DNA结合模块，该结构域在从酵母到人类的所有真核生物中均高度保守。这类具有序列特异性的DNA结合蛋白，最初被鉴定为转录因子，可调控从细胞周期调控到细胞发育命运等多种细胞进程；其表达失调会引发发育缺陷、癌症及衰老相关问题。本团队近期鉴定发现，酿酒酵母的Fkh1与Fkh2蛋白，对于G1期部分复制原点的聚集，以及后续S期内这些原点的提前起始均为必需；这提示二者在连接复制原点的空间结构与其活性之间存在机械调控作用。本研究证实，Fkh1与Fkh2拥有人类FoxP蛋白所特有的结构特征——该特征可使FoxP2与FoxP3形成结构域交换二聚体，能够在体外桥接两个DNA分子。据此，Fkh1在体外与体内均通过依赖保守结构域交换区域的方式发生自身结合，这强烈提示其形成了同源二聚体。Fkh1与Fkh2的结构域交换缺陷（domain-swap-minus, dsm）突变体，仍具备转录因子的功能，但在复制原点的时序调控上存在缺陷。携带dsm突变的Fkh1蛋白可在体内结合复制原点，但无法使其聚集；这一结果支持了如下结论：Fkh1与Fkh2二聚体在染色体元件的空间组织中发挥结构性作用，且该作用具有重要功能意义。