LA JOLLA, Calif.—Embryonic stem cells (ESCs) have long been a point of interest in regenerative medicine, though they lose their pluripotency once they differentiate into different cell types. Thanks to a newly discovered protein complex, Salk Institute researchers recently offered new information in Nature Communications regarding the specifics of how embryonic stem cells are triggered to differentiate--or kept from doing so.
The complex in question is called GBAF. As noted in the recent paper, “To maintain ESC identity, the genome is precisely controlled so that only stem cell-specific transcription programs are turned on while lineage-specific programs are silenced1,2. This control is in part achieved by ATP-dependent chromatin remodeling complexes, which regulate chromatin structure3,4. In particular, several subunits of the mammalian BRG1-associated factors (BAF) chromatin remodeling complex are required for formation of the inner cell mass (ICM) of the embryo and for maintenance of ESCs in vitro5,6,7,8.”
To further explore those subunits, the Salk Institute team—including Diana Hargreaves, an assistant professor in Salk’s Molecular and Cell Biology Laboratory and senior author of the Nature Communications paper—examined BRD9, which has been linked with the BAF family and thought to be one of its subunits. They applied a BRD9 inhibitor to ESCs in dishes and analyzed the cells' pluripotency with regards to any change in activity within the BAF complex.
What they found is that BRD9 works to maintain ESCs in their pluripotent state; when it is inhibited, ESCs begin to move toward differentiation. They also found that rather than just being associated with the BAF family, BRD9 is a component of a then-unidentified BAF complex.
“This project started as an exploration of embryonic stem cell pluripotency, which is this property that allows ESCs to become all different cell types in the body,” said Diana Hargreaves, an assistant professor in Salk’s Molecular and Cell Biology Laboratory and the senior author of the paper. “It’s very important to know how various networks of genes control pluripotency, so finding a previously unknown protein complex that plays such an important regulatory role was very exciting.”
“The genomic binding of the GBAF complex is consistent with its role in maintaining the naive pluripotent state, as inhibition of BRD9 results in transcriptional changes representative of a primed epiblast-like state,” the authors reported in their paper. “Conditional deletion of esBAF subunit ARID1A is not highly correlated with this transition, indicating that GBAF complexes have a functionally specific role in regulating this pathway. We demonstrate that BRD9 is targeted to chromatin via its BD, highlighting the role of this reader domain in GBAF complex targeting.”
They also looked into the question of whether or not BRD9's impact on pluripotency is due to its regulation of gene expression. The team applied the BRD9 inhibitor (I-BRD9) to ESCs and tracked changes in mRNA expression with high-throughput sequencing, and saw “dramatic changes in gene expression.”
“At 24 h, I-BRD9 treatment resulted in 351 differentially expressed genes (DEGs), the vast majority of which were also present among the 929 DEGs changed following 48 h of I-BRD9 treatment (Fig. 1e). In both instances, we observed more downregulated than upregulated genes, suggesting that BRD9 generally maintains gene expression. Gene ontology (GO) analysis of I-BRD9-dependent genes revealed that BRD9 functions primarily in regulating tissue development and cellular differentiation,” the authors explained.
In addition, they noted, their data seemed to indicate that “GBAF complexes may specifically regulate naive pluripotency.”
“What we see with this work is that there’s biochemical diversity at the level of individual variants of the BAF complex that allows for greater regulatory control. Understanding the complexities of that control is going to be key to any regenerative therapies,” said Hargreaves.