Abstract
The asynchronous timing of replication of different chromosome domains is essential for eukaryotic genome stability, but the mechanisms establishing replication timing programs remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under- replication (UR) of late-replicating intercalary heterochromatin (IH) domains in polytene chromosomes1. SUUR negatively regulates DNA replication fork progression across IH; however, its mechanism of action remains obscure2, 3. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)- 67.24, 5. In vitro, Mod(Mdg4) stimulates the ATPase activity of SUUR, although neither SUUR nor SUMM4 can remodel nucleosomes. Mod(Mdg4)-67.2 and SUUR distribution patterns in vivo partially overlap, and Mod(Mdg4) is required for a normal spatiotemporal distribution of SUUR in chromosomes. SUUR and Mod(Mdg4)-67.2 mediate insulator activities of the gypsy mobile element that disrupt enhancer-promoter interactions and establish euchromatin- heterochromatin barriers in the genome. Furthermore, mutations of SuUR or mod(mdg4) reverse the locus-specific UR. These findings reveal that DNA replication can be delayed by a chromatin barrier and thus, uncover a critical role for architectural proteins in replication timing control. They also provide a biochemical link between ATP-dependent motor factors and the activity of insulators in regulation of gene expression and chromatin partitioning.
Competing Interest Statement
Competing interests L.S. and M.C.K. are employed by Epicypher, Inc., a commercial developer and supplier of the EpiDyne nucleosomes and associated remodeling assay platforms used in this study. The remaining authors declare no competing interests.