Fiber-seq reveals the single-molecule architecture of nuclear transcription and the mitochondrial genome

Abstract

Gene regulation is driven by the interplay of numerous features including regulatory factors, genome packaging, and the transcriptional machinery. Established approaches to characterize these features on a genomic scale have largely relied on short-read sequencing, providing an averaged, aggregate view across a large population of cells and obfuscating the underlying single molecule dynamics at play. Meanwhile, complementary single-molecule approaches tend to be limited in scope and scale, even as they provide a more granular view. However, the recent development of long-read single-molecule footprinting approaches like Fiber-seq has provided the potential to bridge the genome-scale breadth of sequencing and the single-molecule resolution of microscopy. In this dissertation, I describe how we applied Fiber-seq to characterize two distinct realms of gene regulation—nuclear transcription and mitochondrial genome organization. First, we used Fiber-seq to visualize RNA polymerases within their native chromatin context at single-molecule and near single-nucleotide resolution along up to 30 kb chromatin fibers. We found that RNA Polymerase II (Pol II) pausing destabilized downstream nucleosomes, with frequently paused genes maintaining a short-term memory of these destabilized nucleosomes. Furthermore, we demonstrated pervasive direct coordination between nearby Pol II genes, Pol III genes, and transcribed enhancers. Overall, we illustrated that transcriptioniv initiation mediates both competition and coordination with nucleosomes and nearby transcriptional machinery along individual chromatin fibers. Second, we used Fiber-seq to measure the packaging of individual full-length mtDNAmolecules at nucleotide resolution, and found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings revealed the primary architecture of mtDNA packaging and regulation in human cells. In combination with our characterization of nuclear transcription, this work demonstrates the capability of Fiber-seq to capture the complexity of gene regulation in distinct cellular contexts.