We report the genome-wide mapping of ORC1 binding sites in mammals, by chromatin immunoprecipitation and parallel sequencing (ChIP-seq). ORC1 binding sites in HeLa cells were validated as active DNA replication origins (ORIs) using Repli-seq, a method that allows identification of ORI-containing regions by parallel sequencing of temporally ordered replicating DNA. ORC1 sites were universally associated with transcription start sites (TSSs) of coding or noncoding RNAs (ncRNAs). Transcription levels at the ORC1 sites directly correlated with replication timing, suggesting the existence of two classes of ORIs: those associated with moderate/high transcription levels ($1 RNA copy/cell), firing in early S and mapping to the TSSs of coding RNAs; and those associated with low transcription levels (<1 RNA copy/cell), firing throughout the entire S and mapping to TSSs of ncRNAs. These findings are compatible with a scenario whereby TSS expression levels influence the efficiency of ORC1 recruitment at G 1 and the probability of firing during S.[Supplemental material is available for this article.] DNA replication is a highly orchestrated process that ensures fidelity of genomes during duplications, as well as their adaptation to variations in cell division, DNA damage, and, in metazoa, chromatin changes associated with development and differentiation. It initiates from multiple chromosomal loci, called replication origins (ORIs), which are selected in the G 1 phase of the cell cycle by sequential recruitment of the origin recognition complex (ORC), CDC6, CDT1, and the MCM complex (the pre-replicative complex; pre-RC). Selected pre-RCs are then sequentially activated during the S phase, following a tight temporally ordered program (Mechali 2010).In Saccharomyces cerevisiae, ORIs contain a 12-bp consensus for ORC binding (Bell and Stillman 1992). Genome-wide analyses of ORIs by chromatin immunoprecipitation and parallel sequencing (ChIP-seq) using anti-ORC or -MCM antibodies showed that this consensus is essential but not sufficient for origin activity and identified other features that influence selection and replication timing, including transcription and/or chromatin structure (Eaton et al. 2010). In metazoa, instead, pre-RC does not exhibit sequence specificity, and the number of potential ORIs is considerably larger, following a process of selection that differs according to cell type, functional status, or stress conditions (Mechali 2010).These further levels of complexity allow DNA replication to adapt to the unique expression patterns of individual cell types. Little is known, however, about the regulation of ORI selection and replication timing in metazoa.
Polycomb (PcG) complexes maintain the silent state of target genes. The mechanism of silencing is not known but has been inferred to involve chromatin packaging to block the access of transcription factors. We have studied the effect of PcG silencing on the hsp26 heat shock promoter. While silencing does decrease the accessibility of some restriction enzyme sites to some extent, it does not prevent the binding of TBP, RNA polymerase, or the heat shock factor to the hsp26 promoter, as shown by chromatin immunoprecipitation. However, we find that in the repressed state, the RNA polymerase cannot initiate transcription. We conclude that, rather than altering chromatin structure to block accessibility, PcG silencing in this construct targets directly the activity of the transcriptional machinery at the promoter.
Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.
Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. We show that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. Our results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. We propose that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator.
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