Technologies for protein and RNA-focused chromosome conformation

If you unpack all of the DNA in a single cell and lay it out flat, it's about six feet long -- and yet it can all fit into a micron-scale nucleus. Not only does it fit, but it's folded within the nucleus precisely to allow for the same DNA sequence across cell types to be specifically accessed for distinct functions. My thesis sought to investigate how DNA is folded within a cell, and how this folding impacts gene regulation and human disease. There are currently two main approaches to measure DNA folding: imaging and chromosome conformation capture (3C) methods. Although imaging is incredibly powerful, it is limited in throughput and resolution. 3C relies on a proximity ligation -- generating a hybrid "contact" fragment of two pieces of DNA that are near each other in 3D space. While there are many 3C-based methods, arguably the most transformative is Hi-C, which applies next-generation sequencing to a 3C library and has identified multiple DNA organizational features within a cell. However, Hi-C is inherently restricted due to sampling space: all possible 3D interactions are being measured. Because of this, billions of sequencing reads are needed to start observing confident DNA interactions over background. To combat this sampling space issue, we developed a method called HiChIP, which combines chromatin immunoprecipitation (ChIP) with Hi-C, allowing for a directed view of long-range contacts associated with a protein factor of interest. HiChIP improves the yield of contact reads by over 10-fold and lowers the input requirement over 100-fold relative to that of previous methods. As a proof of principle, we performed HiChIP on cohesin, a protein complex that has been reported to stabilize chromatin loops. Cohesin HiChIP identified a similar set of loops as Hi-C maps with increased signal-to-background and ten-fold less sequencing. The HiChIP technology we developed allowed, for the first time, DNA folding to be measured in disease-relevant patient samples. Precisely measuring how the immune system operates is central to our understanding of autoimmune disease and cancer. However, due to technological limitations the principles that govern regulatory 3D interactions in disease-relevant patient samples have been incompletely understood. This gap in understanding is particularly problematic for interpreting the molecular functions of inherited risk factors for common human diseases, which reside in intergenic enhancers or other non-coding DNA features in up to 90% of cases. We therefore performed HiChIP on the enhancer and promoter-associated histone mark H3K27ac in sorted T cell subsets and examined DNA interactions to map autoimmune disease variants present in non-coding regions and identify which genes they contact with. We found that the majority of disease-associated enhancers interact with genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for these autoimmune disease SNPs. While protein-factors are well-known to regulate the 3D structure of the genome, less is known regarding RNA. However, specific RNAs, such as XIST and HOTTIP, have been reported to utilize the topology of the genome in order to carry out their functions. We therefore sought to modify the HiChIP method to enrich for DNA interactions focused around an RNA of interest (HiChIRP). Interestingly, we identified many RNAs that engage in different types of 3D interactions. For example, we found that the RNA lncRNA-EPS binds to the boundaries of topological domains, a layer of DNA organization, to then engage in 3D interactions and regulate target genes contained within the domain. In summary, my thesis provided an unprecedented view of the 3D genome through the development of novel genomic tools for the research community. Importantly, these tools enable 3D measurements: (1) efficiently with less sequencing needed, (2) in disease-relevant systems that could not be assayed previously, and (3) focused on regulatory RNAs, whose roles in DNA structure are incompletely understood. I have then applied these tools across many collaborations to better understand the roles of DNA folding in gene regulation and disease.

Keywords: Genetics.
Advisor: Chang Howard Y. (Howard Yuan-Hao)
Greenleaf William James 
Bassik Michael 
Wysocka Joanna