(91f) Genome-Wide Screening of Epigenetic Modifiers during Cell Reprogramming

Lim, B., University of Pennsylvania
A slight mis-interaction of an enhancer-gene pair could give rise to fatal problems, if the gene is expressed in wrong position at a wrong time. To prevent that, chromatins are often divided into “closed” heterochromatin and “open” euchromatin regions, such that cell-type inappropriate genes remain silenced even in the presence of activators. However, heterochromatin causes problems during cell reprogramming. For example, if some of the key neuronal genes are located in the heterochromatin of fibroblast cells, the neuronal genes won’t be activated even in the presence of all the activators, resulting in poor reprogramming efficiency. Using the combination of genome-wide “seq” techniques like ChIP-seq and RNA-seq and quantitative imaging, we sought to elucidate the mechanism of heterochromatin maintenance and characterize the kinetics of gene expression upon depletion of heterochromatin regulators during direct cell reprogramming from fibroblast cells into hepatic cells. We identified ~100 proteins that are closely associated with the maintenance of heterochromatin domain. We found that RNAi knockout of each protein leads to de-repression of key hepatic genes in the presence of hiHep transcription factors that are known to activate hepatic genes. To analyze whether such de-repression of genes is a direct consequence of heterochromatin de-compaction, we used ChIP-seq and immunostaining to visualize H3K9me3 and H3K27me3 marked domain in the genome. We found that H3K9me3 and H3K27me3 marked heterochromatin regulate distinct set of genes. With our imaging and genomics assays, we hope to provide better cell reprogramming strategies to achieve higher efficiency.