Final Report - Hypergravity Effects on Chromatin Conformation and Nuclear Structure in Cultured Cells using High-throughput Sequencing
The conformation of the human genome is known to play an important role in transcriptional control of gene expression. Our aim is to assess whether exposure of cultured human cardiomyocytes to hypergravity can induce changes in chromatin organization as assayed by Hi-C proximity ligation technique. Hi-C is used to analyze chromatin interactions by using formaldehyde to crosslink regions of chromatin that are in close proximity. The DNA is then fragmented using a restriction enzyme and ligated under dilute conditions to favor intramolecular ligation of cross-linked fragments. Finally, the DNA is sequenced, allowing reconstruction of genomic structure. iCell cardiomyocyte cultures will be exposed to 40 g via an engineered device (incu-fuge) that allows for the chemical fixation of cells on a spinning centrifuge. Additionally, we plan on using fluorescence microscopy to analyze the nuclear and actin cytoskeletal conformation of cardiomyocytes post hypergravity exposure. We hypothesize that chromatin will rearrange in hypergravity conditions, both rapidly due to direct mechanical forces, as well as over longer time frames due to changes to structures necessary for transcriptional responses such as the de novo formation of promoter-enhancer loops. A negative result–no change between 1 g and hypergravity conditions–would nevertheless be an important data point in our understanding of the rheology of the nucleus, and how cells and cellular structures respond to different gravity fields. The cellular physiology of hypergravity has clinical relevance for spaceflight, and can further inform our understanding of microgravity physiology.