Refined mechanism of promoter Nucleosome-Depleted Regions resetting after replication

Replication disrupts chromatin organization. Thus, rapid resetting of nucleosome positioning is essential to maintain faithful gene expression. The initial step of this reconfiguration occurs at Nucleosome-Depleted Regions (NDRs). While studies have elucidated the role of Transcription Factors (TFs) and Chromatin Remodelers (CRs) in vitro or in maintaining NDRs in vivo, none has addressed their in vivo function shortly after replication. Through purification of nascent chromatin coupled with yeast genetics, we dissected the choreography of events governing the proper positioning of the -1/+1 nucleosomes flanking promoter NDRs. Our findings reveal that CRs are the primary contributors of -1/+1 repositioning post-replication, with RSC acting upstream of INO80. Surprisingly, while Reb1 and Abf1 TFs are not essential for NDR resetting, they are required for NDR maintenance via the promotion of H3 acetylations. Taken together, we propose a two-step model for NDR resetting in S. cerevisiae: first, CRs alone reset promoter NDRs after replication, while a combination of TFs and CRs is required for subsequent maintenance. Overall design: Nucleosome occupancy was measured by MNase-seq upon nuclear depletion of Abf1, Reb1, Rpo21, Sth1, Ino80 and Rsc3 in mature and nascent chromatin analyzing 'Input' and 'Edu-labeled' samples respectively. Edu labeling efficiency was measured by comparing G1-arrested and S-phase EdU labeled samples. Sth1 binding was measured by ChEC-seq upon nuclear depletion of Ino80. The binding of Ino80 was measured by ChEC-seq upon nuclear depletion of Sth1. The effect of the TSA treatment on nucleosome positioning was measured by MNase-seq upon depletion of Sth1, Ino80 and Reb1. Please note that processed data file generated from both replicates is linked to the corresponding rep1 sample records.