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 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.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation

“…Recent methodologic advances introduced the chromatin in vivo assay (CiA) system, a variation of chemical induced proximity (CIP), as a novel method to investigate the consequences of locally induced alterations of the chromatin landscape after controlled recruitment of an epigenetic effector ( 43 ). CiA has successfully been applied to study the dynamics of heterochromatin formation at the Oct4 locus in mouse embryonic stem cells (mESCs) after the recruitment of HP1α ( 44 ), components of the PRC2 complex ( 45 ) and, combined with a high throughput small molecule library screen, to identify compounds inducing the formation of euchromatin ( 46 ). Additionally, CiA has been used to investigate the opposing effect of the BAF complex on PRC-induced heterochromatin formation, leading to the formation of accessible chromatin ( 47 , 48 ).…”

Application of Small Molecules in the Central Nervous System Direct Neuronal Reprogramming

“…The modulation of epigenetics is temporal and reversible to the unchangeable DNA sequence of the host cells, which is retained during the disease state and age information of the starting cells. Some new techniques, such as high-throughput sequencing analysis, can be utilized to screen pivotal regulators of epigenetics ( Headley et al, 2019 ; Vandana et al, 2021 ).…”

Relationship between lysine methyltransferase levels and heterochromatin gene repression in living cells and in silico

“…Previous studies on these cell lines have detailed the generation of a heterochromatin domain in response to HP1 recruitment, demonstrating silencing of eGFP coupled with accumulation of heterochromatin marks along with reduction of euchromatin marks. Others have used this cell line to identify heterochromatin formation inhibitors in a high-throughput screen ( 52 ) or compounds that facilitate induced pluripotent stem-cell generation ( 53 ) and to determine the role of DNA methylation on epigenetic memory ( 54 ).…”