The influence of RNA:DNA hybrids on chromatin composition, genome structure, and transcription

Abstract

RNA:DNA hybrids are formed when a single-stranded RNA anneals to a complementary strand within a DNA duplex. This results in the displacement of a single-stranded DNA, with the entire nucleic acid structure referred to as an R-loop. In this study, I aimed to characterize RNA:DNA hybrids genome-wide in order to elucidate their role in cell functions. In Chapter 1 of this thesis, I review the current knowledge about non-canonical nucleic acid structures, including RNA:DNA hybrids, and their influence on cellular processes and disease.;RNA:DNA hybrids exist in an intermediate helical structure between an A-form and B-form helix. This conformation is specifically recognized by the S9.6 antibody with little to no sequence specificity. In Chapter 2, I describe the optimization of the genome-wide RNA:DNA hybrid immunoprecipitation (RDIP) assay utilizing the S9.6 antibody to isolate these structures from two human cell lines, IMR-90 and HEK 293T cells. Additionally, this chapter discusses the development of a directional sequencing approach which allowed me to exclusively sequence the RNA strand of the RNA:DNA hybrid. This revealed the RNA component to be purine-rich, indicating a thermodynamic stability contributes to the maintenance of these structures in vivo.;Chapter 2 also details the remainder of our investigation of RNA:DNA hybrids, including subcellular localization studies, enrichment and depletion of co-localization with known genomic features on a local and global scale, influence on gene expression, and the identification of RNA:DNA hybrid binding proteins. These studies revealed RNA:DNA hybrids are locally enriched at repetitive loci, along with areas of decreased methylation and increased DNase hypersensitivity. Additionally I found a larger scale co-localization with heterochromatin associated histone marks. This research also supports a model of RNA:DNA hybrid formation in trans, as opposed to a co-transcriptional formation.;This thesis concludes with Chapter 3, which is a discussion of the implications of these findings and the future of the field studying non-canonical nucleic acid structures.;In summary, this thesis discusses the results of a genome-wide characterization of RNA:DNA hybrids in human cell lines which can be used as the foundation for further studies into their apparent heterogeneous function.