Chemical screen for epigenetic barriers to single allele activation of Oct4.

ABSTRACT

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiAOct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiAOct4 allele contains an engineered EGFP reporter replacing one copy of the Oct4 gene, with an upstream Gal4 array in the promoter that allows recruitment of chromatin modifying machinery. We stimulated gene activation of the CiAOct4 allele by binding a transcriptional activator to the Gal4 array. As with cellular reprograming, this process is inefficient with only a small percentage of the cells re-activating CiAOct4 after weeks. Epigenetic barriers to gene activation potentially come from heavy DNA methylation, histone deacetylation, chromatin compaction, and other posttranslational marks (PTM) at the differentiated CiAOct4 allele in MEFs. Using this platform, we performed a high-throughput chemical screen for compounds that increased the efficiency of activation. We found that Azacytidine and newer generation histone deacetylase (HDAC) inhibitors were the most efficient at facilitating directed transcriptional activation of this allele. We found one hit form our screen, Mocetinostat, improved iPSC generation under transcription factor reprogramming conditions. These results separate individual allele activation from whole cell reprograming and give new insights that will advance tissue engineering.