Mechanisms of p300 Activation During Pluripotency and Differentiation.

SUMMARY
Pluripotency is a critical model for understanding fundamental principles of cell fate specification. This process
makes selective use of enhancers, regulatory elements that facilitate the transcription of cell-type specific
genes. Although changes in enhancer activity are predicted to have broad developmental and pathological
implications, we currently have limited understanding of how enhancers regulate development and disease, in
part due to our incomplete understanding of the mechanisms by which enhancers regulate gene expression.
Increased understanding of the molecular events that regulate enhancer activity, particularly under
developmental contexts, will provide important insights into congenital diseases that occur with their
dysregulation. The chromatin state at enhancers is characterized by the presence of the histone variant, H3.3,
and recruitment of the CBP/p300 family of transcriptional coactivators. Our recent studies demonstrate that
H3.3 deposition at enhancers allows for variant-specific phosphorylation that stimulates p300 acetyltransferase
activity towards its substrate histone H3 lysine 27 (H3K27ac) in mouse embryonic stem cells (ESCs). Further,
we find that CBP and p300 carry out distinct functions in ESCs, with p300 playing a greater role in maintaining
H3K27ac in ESCs. Finally, we find that reduced H3K27ac due to H3.3 or p300 deletion is well tolerated in
ESCs, with little correlated change in transcription. However, both H3.3 and p300 are required for
differentiation, suggesting that H3.3 deposition and subsequent high levels of H3K27ac may be more important
for activating gene transcription than for maintaining ongoing transcription in ESCs. The objective of this
proposal is to dissect the molecular and functional mechanisms by which enhancers are activated both in
pluripotency and differentiation. In the first aim, we will determine how H3.3 phosphorylation stimulates p300
activity in ESCs. In the second aim, we will determine why H3K27 acetyltransferase activity is restricted to
p300 in ESCs. Finally, in the third aim, we will use the tools of chemical biology and novel mouse models that
we have generated to ask how H3.3 and p300 function to promote transcription during pre-implantation
development. Collectively, our work will explore molecular links between H3.3 and p300 in enhancer activation
during the time at which the first lineage specification events occur, with important implications for
understanding how enhancer dysregulation contributes to human disease.

Grant Number: 5R01HD109239-04
NIH Institute/Center: NIH
Principal Investigator: Laura Banaszynski