Studies on the transcriptional regulation of the proneural phox2a gene during neural crest cell development

Neural crest cells (NCCs) differentiate as neurons, glia, melanocytes, and craniofacial-mesenchymal derivatives. This study emphasizes NCCs of the embryonic trunk region, differentiating as melanocytes, sensory neurons, and sympathoadrenal (SA) cells. cAMP and bone morphogenic proteins (BMPs) are crucial signaling molecules for SA cell development. BMPs induce basic helix-loop-helix (bHLH) transcription factor ASH1, inducing homeodomain (HD) transcription factor Phox2a, required for SA cell development. Moderate CAMP signaling, in synergy with BMP2, promotes SA cell development and expression of Phox2a. The mechanism regulating phox2a gene transcription is unknown. These mechanistic studies of phox2a gene regulation tested the hypothesis: Phox2a is directly regulated by Ash1 and cAMP, via Ash1 response element (E-box) and cAMP response element (CRE) on the Phox2a promoter. Employing the human phox2a promoter and its deletions, transient transfections were performed in quail NCCs and mouse CNS-derived catecholaminergic CAD cells. Interestingly, transfected hphox2a promoter activity, in response to cAMP and BMP2 signaling, does not parallel expression patterns and developmental effects of the endogenous phox2a gene. Treatment of NCCs or CAD cells with histone deacetylase (HDAC) inhibitor trichostatin A (TSA), in conjunction with BMP2 and cAMP co-treatment, increases phox2a and TH mRNA expression, and prolongs CREB phosphorylation at ser 133, suggesting a chromatin remodeling mechanism. To demonstrate phox2a transcription mediated by combined BMP2 and cAMP treatment regulates chromatin remodeling, putative CRE half sites were identified within both human and mouse promoter and intron. Chromatin immunoprecipitation assays with CREB, CBP, and acetylated H4 antibodies we demonstrate mouse CRE sites 1 and 2, approximately 5kb upstream from +1, exhibit enhanced CREB/CBP binding with BMP2 and cAMP co-treatment as well as enhanced association with acetylated H4, indicating transcriptionally permissive chromatin. These sites are flanked by E-box cis-acting elements, known binding sites for bHLH proteins like Ash1. Accordingly, I accept my hypothesis and conclude that CRE half-sites 1 and 2 mediate the phox2a transcriptional induction observed with combined BMP2 and cAMP treatment. The adjacent E-boxes suggests their involvement in mediating the BMP2 response, thus offering a mechanistic explanation of the synergistic effect of BMP2 and cAMP signaling on phox2a expression and SA cell differentiation