Project description:We sequenced RNA Pol II Flag pulldowns (NET-seq) from five samples of W303 bar1D (asynchronous, alpha-Factor arrested, nocodazole arrested or transformed with a pGAL1 multicopy plasmid carrying either no ORF or the SEN1 ORF)
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:Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here, we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, NTP substrate levels, and RNA polymerase II (Pol II) activity to TSS selection by "promoter scanning" in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1, and +1 positions quantitatively in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to GTP levels at the first five transcript positions and to CTP and UTP levels at the second position genome wide. These results suggest individual NTP levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.
Project description:We provide the genome-wide map of Esrrb binding in mitotic and asynchronous ES cells together with the Esrrb-induced transcriptomic changes in early G1, late G1 and G2.
Project description:Chromatin is highly condensed and transcriptionally repressed during mitosis. Although it is established that some general transcription factors are inactivated by phosphorylation at mitosis, many details of mitotic transcriptional repression and its underlying mechanisms are largely unknown. Here, we provide evidence that as cells enter mitosis, genes with transcriptionally engaged RNA Polymerase II (Pol II) can continue transcription until the end of the gene to clear Pol II from mitotic chromatin. Using ChIP-Seq, we find that the transcriptional reinitiation process is globally impaired in early mitosis (prophase/prometaphase), with loss of TFIIB occupancy and nucleosome-free regions at promoters. Pretreatment of nocodazole-arrested mitotic cells with the P-TEFb inhibitor flavopiridol prevents the release of promoter-proximal engaged Pol II. Global nascent RNA sequencing and RNA fluorescence in situ hybridization (FISH) of individual genes demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Chemical and mutational inhibition of P-TEFb in mitosis leads to delays in the progression of cell division. Together, our study reveals a novel mechanism for mitotic transcriptional repression whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. ChIP-Seq of Pol II of different forms, TFIIB, H3K4me3 in human HeLa cells at different cell cycle stages. ChIP-Seq of Pol II in HeLa mitotic cells with or without CDK9 inhibitor flavopiridol pretreatment. Nascent RNA-seq in asynchronous and arrested mitotic cells.