The cAMP pathway in combination with BMP2 regulates Phox2a transcription via cAMP response element binding sites.
Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor Phox2a, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on Phox2a transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPhox2a-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous Phox2a expression, the 7.5-kb hPhox2a reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPhox2a fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPhox2a promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous Phox2a transcription and prolonged CREB phosphorylation, indicating Phox2a chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine Phox2a promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to Phox2a-luciferase reporters. We conclude these CREs are functional, with CREB directly activating Phox2a transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on Phox2a transcription.
Benjanirut
Chutamas
C
Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Paris
Maryline
M
Wang
Wen-Horng
WH
Hong
Seok Jong
SJ
Kim
Kwang Soo
KS
Hullinger
Ronald L
RL
Andrisani
Ourania M
OM
D000818:Animals; D001665:Binding Sites; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D050882:CREB-Binding Protein; D002843:Chromatin; D000242:Cyclic AMP / Q000378:metabolism* / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000502:physiology*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000502:physiology*
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D002843:Chromatin; D018398:Homeodomain Proteins; C491060:Phox2a protein, mouse; D016212:Transforming Growth Factor beta; D000242:Cyclic AMP; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse