(486c) Deep Profiling of Heterochromatin Associated Proteins and Their Role in Liver Cell Reprogramming

At a given time in development, each cell has to regulate activation of tens to hundreds of genes to ensure normal development. To facilitate robust gene control, a bigger layer of regulation is in play by dividing each chromosome into active euchromatin and inactive heterochromatin regions. Despite the presence of activators, genes located in the heterochromatin region are silenced, restraining the domain where a cell has to regulate at a given time. While the formation of heterochromatin helps reduce ectopic activation of genes, this poses a problem during stem cell reprogramming. Indeed, addition of hiHep factors, which should direct stem cells to differentiate into liver cells, often fails because many of the hepatic genes located in the compacted heterochromatin domain stay silenced. Using the combination of genome-wide “seq” techniques and quantitative imaging, we identified proteins that are responsible for the heterochromatin formation in mammalian fibroblast cells, and analyzed how they affect different aspect of heterochromatin.

In order to screen for proteins that are responsible for the formation of heterochromatin, we treated fibroblast cells with siRNAs targeting each of the candidate protein, and examined the change in expression level of key hepatic genes such as DSC2, NR1H4, and CRP. Using RNA-seq, we identified 90 proteins that led to overexpression of the hepatic genes. To further understand how each candidate protein affects the formation of heterochromatin, we stained the siRNA treated cells with H3K9me3 antibody which works as a marker for heterochromatin domain and analyzed the change in heterochromatin structure in a single cell resolution. We examined how diffuse and moderately compacted heterochromatin region and highly compacted focal region of heterochromatin are affected upon different siRNA treatment. Some siRNA-treated cells exhibited both fewer densely packed and moderately packed chromatin regions. Some other siRNA-treated cells showed changes in either focal heterochromatin regions or diffuse regions, but not both. Our results imply that heterochromatin proteins regulate heterochromatin formation and subsequent gene silencing in a differential manner. This study will provide better understanding on the mechanism of gene silencing via heterochromatin formation, as well as to suggest better stem cell reprogramming strategies.