PDF(Pubmed)
Abstract：
In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins\' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.
摘要：
在酿酒酵母中，叉头（Fkh）转录因子Fkh1（叉头同源物）增强了许多在S期早期（早期起源）起作用的DNA复制起点的活性。目前的模型认为，Fkh1通过与邻近起源的Fkh1结合位点（FKH位点）结合而直接促进这些起源的活性。然而,Fkh1用于促进早期起源活性的后DNA结合功能知之甚少。Fkh1包含一个保守的FHA（叉头关联）域，对含磷酸苏氨酸(pT)的配偶体蛋白具有特异性的蛋白质结合模块。在一小部分酵母起源中，Fkh1-FHA结构域增强了ORC（起源识别复合物）-起源结合步骤，启动原点循环的G1阶段事件。然而,Fkh1-FHA结构域在基因组规模上对染色体复制或ORC-起点相互作用的重要性尚不清楚.这里,S期SortSeq实验用于比较增殖的FKH1和fkh1-R80A突变细胞中的基因组复制。Fkh1-FHA结构域促进了约100个起源的活性，这些起源在早期到中期S期起作用，包括大多数与着丝粒相关的起源，同时抑制≈100个晚期起源。因此，在没有功能性Fkh1-FHA结构域的情况下，酵母基因组的时间景观变平了。起源与定位的核小体阵列相关，该核小体阵列在起源上框住核小体耗尽区（NDR），和ORC起源结合对于这种染色质组织是必需的，但不是足够的。为了询问Fkh1-FHA结构域是否对起源的染色质结构有影响，评估从增殖细胞产生的ORC-ChIPSeq数据和从G1-阻滞和增殖的细胞群体产生的MNaseSeq数据。受Fkh1-FHA结构域差异调节的起源组的特征在于该结构域对ORC起源结合和G1期染色质的不同影响。因此，Fkh1-FHA结构域在G1期的早期起源控制着独特的染色质结构，在S期调控着起源活性。
