<p><p>Eukaryotic genomes have extensive flexibility and plasticity to modify transcription and replication programs, yielding a myriad of differentiated cell types and survival mechanisms to adverse environmental conditions. As these genomic processes require precise localization of DNA-binding factors, their dynamic temporal and spatial distributions provide dramatically different interpretations of a static genome sequence. DNA-binding factors must compete with nucleosomes, the basic subunit of chromatin, for access to the underlying DNA sequence. Even though the spatial preferences of these proteins are partially explained by DNA sequence alone, the complete genome occupancy profile has remained elusive, and we currently have a limited understanding of how DNA-binding protein configurations directly impact transcription and replication function.</p></p><p><p>Profiling the entire chromatin environment has typically required multiple experiments to capture both DNA-binding factors and nucleosomes. Here, we have extended the traditional micrococcal nuclease (MNase) digestion assay to simultaneously resolve both nucleosomes and smaller DNA-binding footprints in <i>Saccharomyces cerevisiae</i>. Visualization of protected DNA fragments revealed a nucleotide-resolution view of the chromatin architecture at individual genomic loci. We show that different MNase digestion times can capture nucleosomes partially unwrapped or complexed with chromatin remodelers. Stereotypical DNA-binding footprints are evident across all promoters, even at low-transcribed and silent genes. By aggregating the chromatin profiles across transcription-factor--binding sites, we precisely resolve protein footprints, yielding <i>in vivo</i> insights into protein-DNA interactions. Together, our MNase method, in one experiment, provides an unprecedented assessment of the entire chromatin structure genome-wide.</p></p><p><p>We utilized this approach to interrogate how the replication program is regulated by the chromatin environment surrounding DNA replication initiation sites. Pre-replicative complex (pre-RC) formation commences with recruitment of the origin recognition complex (ORC) to specific locations in the genome, termed replication origins. Although successful pre-RC assembly primes each site for S-phase initiation by loading the Mcm2-7 helicase, replication origins have substantially different activation times and efficiencies. We posited that replication origin function is substantially impacted by the local chromatin environment. Here, we resolved a high-resolution ORC-dependent footprint at 269 replication origins genome-wide. Even though ORC in <i>S. cerevisiae</i> remains bound at replication origins throughout the cell cycle, we detected a subset of inefficient origins that did not yield a footprint until G1, suggesting a more transient ORC interaction prior to pre-RC assembly. Nucleosome movement accommodated the pre-RC-induced expansion of the ORC-dependent footprint in G1, leading to increased activation efficiency. Mcm2-7 loading is preferentially directed to one side of each replication origin, in close proximity to the origin-flanking nucleosome. Our data demonstrates that pre-RC components are assembled into multiple configurations <i>in vivo</i>.</p></p><p><p>We anticipate that extending chromatin occupancy profiling to many different cell types will reveal further insights into genome regulation.</p></p> / Dissertation
Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/11371 |
Date | January 2015 |
Creators | Belsky, Jason Alan |
Contributors | MacAlpine, David M |
Source Sets | Duke University |
Detected Language | English |
Type | Dissertation |
Page generated in 0.0103 seconds