DNA replication requires the recruitment of a pre-replication complex facilitated by Origin Recognition Complex (ORC) onto the chromatin during G1 phase of the cell cycle. The ORC-associated protein (ORCA/LRWD1) stabilizes ORC on chromatin. Here, we evaluated the genome-wide distribution of ORCA using ChIP-seq during specific time points of G1. ORCA binding sites on the G1 chromatin are dynamic and temporally regulated. ORCA association to specific genomic sites decreases as the cells progressed towards S-phase. The majority of the ORCA-bound sites represent replication origins that also associate with the repressive chromatin marks H3K9me3 and methylated-CpGs, consistent with ORCA-bound origins initiating DNA replication late in S-phase. Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. Regions that associate with both ORCA and H3K9me3, exhibit diminished H3K9 methylation in ORCA-depleted cells, suggesting a role for ORCA in recruiting the H3K9me3 mark at certain genomic loci. Similarly, DNA methylation is altered at ORCA-occupied sites in cells lacking ORCA. Furthermore, repressive chromatin marks influence ORCA's binding on chromatin. We propose that ORCA coordinates with the histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of origins, which primes them for late replication.

Significance: Ubiquitination of several DNA replication–repair proteins is a critical mechanism by which cellular DNA replication and DNA damage repair pathways are controlled. The E3 ligase RING finger and WD repeat domain-containing protein 3 (RFWD3) has emerged as an important regulator of genome stability. We propose that the association of RFWD3 to proliferating cell nuclear antigen stabilizes RFWD3 at the fork, enabling ubiquitination of replication protein A and its subsequent removal to facilitate DNA replication.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Significance: Origin recognition complex (ORC) is required for the initiation of DNA replication. Unlike other ORC components, the role of human Orc6 in replication remains to be resolved. We identified an unexpected role for hOrc6, which is to promote S-phase progression after prereplication complex assembly and DNA damage response. Orc6 localizes at the replication fork, is an accessory factor of the mismatch repair complex, and plays a fundamental role in genome surveillance during S phase.

BEN domain–containing proteins are emerging rapidly as an important class of factors involved in modulating gene expression, yet the molecular basis of how they regulate chromatin function and transcription remains to be established. BEND3 is a quadruple BEN domain–containing protein that associates with heterochromatin and functions as a transcriptional repressor. We find that BEND3 is highly expressed in pluripotent cells, and the induction of differentiation results in the down-regulation of BEND3. The removal of BEND3 from pluripotent cells results in cells exhibiting upregulation of the differentiation-inducing gene expression signature. We find that BEND3 binds to the promoters of differentiation-associated factors and key cell cycle regulators, including CDKN1A, encoding the cell cycle inhibitor p21, and represses the expression of differentiation-associated genes by enhancing H3K27me3 decoration at these promoters. Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation. Significance The molecular basis of how the BEN domain–containing gene family regulates chromatin function and transcription remains to be elucidated. We report that BEND3 is highly expressed in pluripotent cells and binds to promoters of genes involved in differentiation. BEND3 regulates the expression of differentiation-associated genes by modulating the chromatin architecture at promoters. We propose that transcription repression mediated by BEND3 is essential for normal development and maintenance of pluripotency.