In a single human cell, two meters of DNA is carefully packaged within a five-micron nucleus in such a way that allows complex biological necessities such as genome replication, DNA repair, and regulated gene expression. This spectacular organizational challenge is overcome through the hierarchical folding of DNA into chromatin. Our understanding of the structure of chromatin, from the level of individual nucleosomes at the ~100 bp level to higher-order, long-range interactions of chromosomes at the megabase level, is undergoing a profound expansion due to high-throughput methods of mapping structural elements to specific locations on the genome, allowing correlation with biological state. We expect the structure of chromatin at an intermediate length scale of ~2 kilobases to play a crucial role in regulating transcription, DNA replication, and DNA repair, but our structural understanding of this "secondary structure" of chromatin organization continues to lag behind our rapidly developing understanding of both the level of nucleosomes and higher-order, long-range interactions. After decades of work on the intermediate level of chromatin, the topology of this structure - or indeed the very existence of a well-stereotyped structure in vivo - is still holy debated, and almost nothing is known about the variability of these putative structures as a function of genome position. This pilot project builds methods that will develop a clearer picture of this scale of chromatin organization in order to integrate both the physical and biochemical views of the nucleus. We will study chromatin folding both in vivo and in vitro by applying ionizing radiation, which is known to generate correlated nicks to the DNA backbone at spatially proximal locations. The resulting single-stranded DNA fragments, which have ends that were within ~3 nm of each other in the folded chromatin structure, will be analyzed with high-throughput sequencing in order to map these fragments to the genome. This analysis will generate genome-wide pairwise distance constraints on the folded DNA. These data will provide an entirely new window into chromatin compaction and structure at the 30- nm length scale. Our investigations will begin with chromatin fibers assembled in vitro in order to troubleshoot and validate our methodology. Next we will investigate chromatin structure in S. cerevisiae, a model system with a small genome and extremely well characterized, well-positioned nucleosomes. Finally, we will pilot our chromatin structure mapping methodology in primary human fibroblasts and immortalized B-cells. This structural information will be combined with and compared to existing data sets that describe chromatin modifications and nuclease accessibility, laying the groundwork for an integrated physical model of chromatin structure by bridging the gap between our crystallographic understanding of the mononucleosome and our emerging understanding of the higher-order interactions at the megabase scale.
Genomic structure is beginning to be understood at length scales of millions of bases of DNA as well as in the range of hundreds of bases of DNA, but there remains a gap in our understanding at length scales in the thousands. We propose a new, powerful method that harnesses ionizing radiation and high-throughput sequencing to map the genomic architecture at this intermediate length scale. A better understanding of the structure of the human genome will help to shed light on basic biological process, such as gene expression, that are fundamental to our understanding of human health and disease.
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