Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Genet. 2022 Jul 6;18(7):e1010277. doi: 10.1371/journal.pgen.1010277. eCollection 2022 Jul.
The nucleosome remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants depleted of the catalytic subunits of these CRs for binding of transcriptional activator Gcn4 and recruitment of TATA-binding protein (TBP) during preinitiation complex (PIC) assembly. RSC and Ino80 were found to enhance Gcn4 binding to both UAS elements in NDRs upstream of promoters and to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at many UAS elements, indicating a dual mode of action that is modulated by the presence of RSC. RSC and SWI/SNF collaborate to enhance TBP recruitment at Gcn4 target genes, together with Ino80C, in a manner associated with nucleosome eviction at the TBP binding sites. Cooperation among the CRs in TBP recruitment is also evident at the highly transcribed ribosomal protein genes, while RSC and Ino80C act more broadly than SWI/SNF at the majority of other constitutively expressed genes to stimulate this step in PIC assembly. Our findings indicate a complex interplay among the CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly.
核小体重塑复合物（CR）SWI/SNF、RSC 和 Ino80C 在移除或重新定位核小体方面合作，以在氨基酸饥饿诱导的许多酵母基因的启动子处产生核小体耗尽区域（NDR）。我们分析了这些 CR 的催化亚基缺失突变体，以研究在起始前复合物（PIC）组装过程中，转录激活因子 Gcn4 的结合和 TATA 结合蛋白（TBP）的募集情况。发现 RSC 和 Ino80C 增强了 Gcn4 与启动子上游 NDR 中 UAS 元件的结合，以及核小体占据的编码序列内的非常规结合位点的结合；当 RSC 耗尽时，SWI/SNF 有助于 UAS 结合。所有三种 CR 都被 Gcn4 主动募集到大多数 UAS 元件，并且似乎通过降低结合基序处的核小体占有率来增强 Gcn4 结合，表明存在正调控环。出乎意料的是，SWI/SNF 在 WT 细胞中作用是防止许多 UAS 元件的 Gcn4 过度结合，表明存在一种由 RSC 调节的双重作用模式。RSC 和 SWI/SNF 共同作用，与 Ino80C 一起，增强 Gcn4 靶基因的 TBP 募集，同时在 TBP 结合位点处伴随着核小体的逐出。在高度转录的核糖体蛋白基因中，CR 之间的合作也很明显，而 RSC 和 Ino80C 在大多数其他组成型表达基因中的作用比 SWI/SNF 更广泛，以刺激 PIC 组装的这一步骤。我们的研究结果表明，CR 之间存在复杂的相互作用，以逐出启动子核小体来调节激活剂结合并刺激 PIC 组装。
