Chemical screen for epigenetic barriers to single allele activation of Oct4

Kathryn M Headley,Katarzyna M Kedziora,Aidin Alejo,Elianna Zhi-Xiang Lai,Jeremy E Purvis,Nathaniel A Hathaway

doi:10.1016/j.scr.2019.101470

Kathryn M Headley, Katarzyna M Kedziora + Show 4 more

Open Access

https://doi.org/10.1016/j.scr.2019.101470

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Abstract

Here we utilized the chromatin in vivo assay (CiA) mouse platform to directly examine the epigenetic barriers impeding the activation of the CiA:Oct4 allele in mouse embryonic fibroblasts (MEF)s when stimulated with a transcription factor. The CiA:Oct4 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 CiA:Oct4 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 CiA:Oct4 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 CiA:Oct4 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.

Highlights

Regenerative medicine aims to replace damaged tissues with healthy engineered tissues (Tian et al, 2012; Walia et al, 2012)
Our results provide for a robust epigenetic screen for endogenous single allele Oct4 activation chemical enhancers combining a directed transcription factor and small molecule
We found that histone deacetylase (HDAC) inhibition seemed to result in primary peak activation occurring by 30 h while DNMT inhibition resulted in gradual activation with peak activation by hour 60

Summary

Introduction

Regenerative medicine aims to replace damaged tissues with healthy engineered tissues (Tian et al, 2012; Walia et al, 2012). Many current regenerative medicine techniques use human derived stem cells (hESCs) from a donor to regenerate damaged tissues upon stem cell injection or to regenerate tissues in vitro which can be transplanted into the patient (Bongso and Richards, 2004; Mao and Mooney, 2015; Olson et al, 2011). Recent regenerative medicine research has found methods to efficiently generate safer iPSCs (Attwood and Edel, 2019; Cyranoski, 2018; Feng et al, 2009; Li and Li, 2014; Sanal, 2014; Sharma, 2016) Some of these techniques include small molecule facilitation of induced reprogramming which have resulted in more efficient cellular reprograming (Feng et al, 2009; Ichida et al, 2009; Li et al, 2011; Nie et al, 2012; Shi et al, 2008; Yuan et al, 2011; Zhu et al, 2010)

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