Project description:Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae (HTS)
Project description:The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1-phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization. These files contain BrdU-IP-seq files (2 replicates for each strain; 5 strains total where WT was used as control). These files also contain RNA-seq data from Asynchronous and G1 arrested WT and Mutant cells (2 replicates per strain and condition; paired end). These files also contain RNA-pol-chip-seq data from Asynchronous and G1 arrested WT and Mutant cells (2 replicates per strain/condition).
Project description:Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating matingtype silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycleregulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome. 30 total ChIP-chip samples were analyzed in triplicate. Two antibodies were used, and samples lacking epitopes were processed as controls.
Project description:The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1-phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.
Project description:The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1-phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.
Project description:Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified S. cerevisiae Fkh1 and Fkh2 as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homo-dimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.
Project description:Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified S. cerevisiae Fkh1 and Fkh2 as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homo-dimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.
Project description:Extensive changes in replication timing occur during early mouse development, but their biological significance remains uncertain. To identify evolutionarily conserved features of replication timing and their relationships to epigenetic properties in humans, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two independently derived human induced pluripotent stem cell lines (hiPSCs). Results confirm the conservation of coordinately replicated megabase-sized units of chromosomes (replication domains) with stable cell type specific molecular boundaries that consolidate into larger replication domains during differentiation. Replication timing changes encompassed units of 400-800 kb and were coordinated with changes in transcription similar to mouse. Moreover, significant cell-type specific conservation of replication timing profiles was observed across regions of conserved synteny, despite significant species variation in the alignment of replication timing to isochore GC/LINE-1 content. Replication profiling also revealed a closer genome-wide epigenetic alignment of hESCs to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Finally, we identify a signature of chromatin modifications marking the boundaries of early replicating domains and a remarkably strong link between spatial proximity of chromatin as measured by Hi-C analysis and replication timing. Together, our results reveal evolutionarily conserved elements of the replication program in mammalian early development, demonstrate the power of replication profiling to identify important epigenetic distinctions between closely related stem cell populations (e.g. ESCs vs. EpiSCs), and strengthen the hypothesis that replication domains are structural and functional units of 3D chromosomal architecture. 8 cell types, with a total of 13 individual replicates (i.e. 5 in duplicates, 3 in single replicates)
Project description:Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating matingtype silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycleregulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome.