HES1 cooperates with pRb to activate RUNX2-dependent transcription.
The retinoblastoma protein, pRb, can activate the transcription factor RUNX2, an essential regulator of osteogenic differentiation, but the mechanism of this activation is unknown. Here we studied the interaction of pRb and RUNX2 with HES1, previously reported to augment RUNX2 activity. PRb can act to promote RUNX2/HES1 association with concomitant promoter occupancy and transcriptional activation in bone cells.


BACKGROUND
RUNX2 (also known as OSF2/CBFA1) is a transcription factor required for osteoblast differentiation and bone formation. We have reported that RUNX2 can associate with the retinoblastoma protein pRb, a common tumor suppressor in bone, and the resultant complex can bind and activate transcription from bone-specific promoters. This activity of the pRb/RUNX2 complex may thus link differentiation control with tumor suppressor activity. However, the mechanism through which pRb can activate RUNX2 is unknown. HES1 is a reported co-activator of RUNX2 that shares a binding site on RUNX2 with pRb. Thus, we have tested the cooperativity among these factors in activating transcription from bone specific promoters.


METHODS
Coimmunoprecipitation, chromatin immunoprecipitation, and EMSA experiments were used to study the interaction of RUNX2, HES1, and pRb in cell lysates and on DNA. Transcriptional reporter assays were used to analyze the activity of RUNX2 in the presence and absence of HES1 and pRb.


RESULTS
We showed that pRb can associate with HES1, a previously described RUNX2 interactor that can itself augment RUNX2-dependent transcription. The association of HES1 with RUNX2 is augmented by pRb. Furthermore, both pRb and HES1 increase the amount of RUNX2 bound to promoter sites in vivo, pRb and HES1 synergistically activate a RUNX2-dependent reporter gene, and depletion of HES1 reduces RUNX2/pRb activity.


CONCLUSIONS
These data indicate that pRb acts as a RUNX2 co-activator at least in part by recruiting HES1 into the pRb/RUNX2 complex and further elucidate a novel role for pRb as a transcriptional co-activator in osteogenesis.
Lee
Jong-Seo
JS
Department of Pathology, Harvard Medical School Boston, Massachusetts, USA.
Thomas
David M
DM
Gutierrez
Gabriel
G
Carty
Shannon A
SA
Yanagawa
Shin-ichi
S
Hinds
Philip W
PW
D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016160:Retinoblastoma Protein / Q000378:metabolism* / Q000494:pharmacology; D000072056:Transcription Factor HES-1; D015533:Transcriptional Activation
D051792:Basic Helix-Loop-Helix Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D018398:Homeodomain Proteins; D015675:Osteocalcin; D016160:Retinoblastoma Protein; D000072056:Transcription Factor HES-1; C078493:HES1 protein, human