Jefferson Cancer Institute and Departments of Microbiology/Immunology and Pathology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
 

Abstract

The human p300 protein is a cellular target of the adenoviral E1A oncoprotein (Moran 1993 Curr Opin Genet. Dev 3:63).  The region of E1A interacting with p300 is distinct from that interacting with tumor suppressor retinoblastoma protein (Rb) and Rb-related proteins.  The interactions of E1A with both p300 and Rb-family proteins are required for cellular transformation.  The p300 protein is thought a growth supressor and stimulator for differentiation.  It is expressed ubiquitously and its sequence conserved from nematode to human (Arany et al. 1994 Cell 77:799) further implying its important biological role.
 The sequence of p300 reveals it as a potential transcriptional coactivator (Eckner et al. 1994 Genes Dev 8:869).  This prediction is consistent with the facts that p300 has transcriptional activity (Arany et al. 1995 1995 Nature 374:81) and forms a complex with TBP in vivo (Abraham et al. 1993 Oncogene 8:1639).  Further, p300, like the homologous CREB-binding protein (CBP), modulates the CREB transactivation (Lundblad et al. 1995 Nature 374:85).  However, the actual role and significance of this p300 function in cellular control and E1A transformation is unclear.  For transformation, E1A represses skeletal muscle differentiation and transactivation by myogenic regulators, MyoD-family transactivators (Lassar et al. 1994 Curr Opin Cell Biol 6:788).  Thus, p300 is, possibly, involved in transactivation by MyoD-family proteins in the stimulation of muscle differentiation.
 To probe this hypothesis, we investigated the role of p300 in MyoD transactivation by using an E box (MyoD consensus) reporter system in U-2 OS osteosarcoma cells.  In this system MyoD transactivates the reporter but p300 does not without MyoD.  E1A 12S represses this transactivation.  The results obtained from analyses with an E1A 12S deletion of the N terminal p300-binding region and a p300 deletion of the E1A-binding region suggest that the efficient repression by E1A requires its interaction with p300 and p300 positively participates in MyoD transactivation.
 Further, we detected an interaction between p300 and MyoD in vivo and in vitro.  In vitro binding assays with GST fusion proteins mapped the region of p300 interacting with MyoD.  This region locates at the C terminal cysteine/histidine-rich domain and is separated from the CREB binding region at the N terminus.
 To investigate how p300 communicates the basal transcriptional machinery, we also detected its interactions with TBP in vivo and in vitro and with TFIIB in vitro.  By using Gal4-p300 fusion and deletion analysis, we mapped two separate transactivation domains of p300, one at C terminus and the other at N terminus.  Each of them alone induces Gal4 reporter activity over six folds higher than the wildtype.  The C terminal transactivation domain is able to interact with TBP and TFIIB in vitro.  The communication of the N terminal domain with the basal complex is probably mediated by other transcription factors.
 Consistent with its role as a coactivator, ectopic p300 potentiates the MyoD transactivation.  To confirm this function of p300, we made and identified p300 mutants, both dominant positives and dominant negatives that markedly effect endogenous p300 coactivator function in MyoD transactivation.  In this mutation analysis, we observed that the modulation of MyoD transactivation by p300 could be either direct through the interaction with MyoD or indirect through mediation by other factors.  The effect of these p300 mutants on the E box reporter activity were also observed under differentiation condition in C2C12 myoblast and human rhabdomyosarcoma cells.  This function of p300 indicates one of mechanisms that p300 stimulates muscle differentiation and serves as a sensitive point that could be attacked by E1A during transformation.