Summary DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are poorly understood. A role for three-dimensional chromatin organization has been proposed. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells Rif1 coats late replicating domains and, together with Lamin B1 identifies the majority of the late replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during G1, thereby revealing a novel function of Rif1 as organizer of nuclear architecture. Loss of Rif1 affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during S-phase Rif1 ensures temporal coordination of replication of interacting domains. In summary our study identifies Rif1 as the first molecular link between nuclear architecture organization and replication-timing establishment in mammals.

SummaryChromatin remodeling complexes play essential roles in metazoan development through widespread control of gene expression, but the precise molecular mechanisms by which they do this in vivo remain ill defined. Using an inducible system with fine temporal resolution, we show that the nucleosome remodeling and deacetylation (NuRD) complex controls chromatin architecture and the protein binding repertoire at regulatory regions during cell state transitions. This is primarily exerted through its nucleosome remodeling activity while deacetylation at H3K27 follows changes in gene expression. Additionally, NuRD activity influences association of RNA polymerase II at transcription start sites and subsequent nascent transcript production, thereby guiding the establishment of lineage-appropriate transcriptional programs. These findings provide a detailed molecular picture of genome-wide modulation of lineage-specific transcription by an essential chromatin remodeling complex as well as insight into the orchestration of molecular events involved in transcriptional transitions in vivo.Video Abstract