A study published in Nucleic Acids Research and by PAN Guangjin’s group from the Guangzhou Institutes of Biomedicine and Health (GIBH) of the Chinese Academy of Sciences uncovered the function and molecular mechanism of acetyltransferase histone acetyltransferase binding to ORC1 (HBO1) in the maintenance of human embryonic stem cell pluripotency and mesendoderm specification.

Transforming growth factor beta (TGF-β) signaling is a conserved morphogen signal that controls cell fate during embryonic development and adulthood. In particular, TGF-β signaling regulates stem cell maintenance, proliferation, embryonic lineage.  

SMAD Family Member 4 (SMAD4) plays a crucial role in TGF-β signaling in the nucleus. Chromatin related factors (CRFs) have been reported to associate with SMADs and potentialize SMAD functions. However, most of these reported SMAD associated CRFs are not known as lineage specific factors that specify a particular lineage fate. In the meantime, the exact mechanism by which TGF-β family signaling is still unknown. 

In this study, the researchers found that HBO1-/- hESCs fail to maintain pluripotency and spontaneously differentiate into neuroectoderm. They also found that HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. These results suggested that HBO1 plays a critical role to specify mesendoderm cell fate.  

To analyze the function of HBO1, the researchers designed lenti-viral based expression vector of different mutant forms of HBO1 deleted by either NTD or MYST domain, and found that the C terminal MYST acetyltransferase domain is essential to HBO1 functions in hESCs.  

Since HBO1 acetylates histones H3 and H4, the researchers examined H3 or H4 histone acetylation, and showed that H3K14ac while not other histone acetylation is impaired HBO1 deficient cells. They noticed that HBO1 binding peaks greatly co-localized with H3K14ac perks on chromatin by CUT&Tag-seq assay, and the SMAD motif is among the top enriched motifs in HBO1 peak regions. Indeed, HBO1 and SMAD4 were associated based on Co-IP assay. 

Besides, the researchers discovered that the expression of downstream target genes of the TGFβ signaling pathway is significantly reduced when acetyltransferase HBO1 is deleted in the presence of TGFβ. They found that HBO1 is necessary for these gene expression occurring downstream of the TGFβ signaling. The results of CUT&Tag-seq tests showed that SMAD4 chromatin occupancy is not significantly impaired in HBO1-/- hESCs compared with WT hESCs, and HBO1 chromatin enrichment is significantly reduced in 2i (SB431542/Dorsomorphin, inhibitor of the TGFβ signaling pathway) treated hESCs.  

In summary, these findings indicated that HBO1 plays an important role in regulating critical lineage decision in early embryonic development. This study showed the biological function of chromatin remodeling factors regulating cell fate transformation, and offers a novel approach to effectively obtaining the neural lineage cells' technical system.