Origin firing at repetitive sequences and genome replication - Admin Supplement

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Abstract of the Parent Grant (R01 GM117446) Over half of the human genome is comprised of repetitive DNA sequences organized as gene-poor, late replicating, transcriptionally silent heterochromatin. Recent studies have discerned widespread transcriptional de-repression at repeated regions during carcinogenesis and aging. De-repression accelerates replication of these regions and thus compromises replication in the gene-rich transcriptionally active chromatin. Despite the importance and prevalence of the association between low levels of transcription and late replication, the mechanistic basis for this link remains unclear. The ribosomal DNA (rDNA) as well as copper-inducible CUP1 arrays in budding yeast are ideal experimental systems in which to elucidate these mechanisms for two main reasons. First, at each locus, a single manipulation, Sir2 depletion at the rDNA, and cooper administration at the CUP1, activates both transcription and replication, providing a simple tool to manipulate both processes. Second, each rDNA and CUP1 repeat features a single, sequence-defined origin of replication, which creates uniform and predictable positioning of replication initiation factors and nucleosomes in their vicinity. This feature makes these systems, and yeast origins in general, ideal for the application of deep sequencing methods for epigenome profiling we have developed, offering a tremendous advantage over mammalian origins whose lack of sequence-specificity confounds modern deep sequencing-based approaches. Our methods resolves at nucleotide level the precise location of nucleosomes and pre- replicative complexes (pre-RC) at the repetitive as well as in unique regions of the genome. Using these methods, we discovered that transcriptional activation at both rDNA and CUP1 origins leads to reduced occupancy of nucleosomes adjacent to pre- RC, though by different means at the two loci: At CUP1, transcription reduces nucleosome occupancy next to the pre-RC loading site, whereas at the rDNA the nucleosome occupancy next to the pre-RC loading site does not change; instead, RNA Pol-II pushes pre-RC to an adjacent region with low nucleosome density. Using this experimental system and the tools for chromatin profiling we have developed, we will determine whether high nucleosome occupancy adjacent to pre-RC inhibits replication initiation and whether chromatin compaction-mediated inhibition of nucleosome remodelers enforce late replication in transcriptionally silent chromatin.