Abstract
The noradrenergic cell type is characterized by the expression of proteins involved in the biosynthesis, transport, and secretion of noradrenaline and is dependent on the sequential and combinatorial expression of numerous transcription factors, including Phox2a, Phox2b, dHAND, GATA2, GATA3, and MASH1. Phox2a and Phox2b transactivate the promoter of the gene encoding the noradrenergic biosynthetic enzyme, dopamine β-hydroxylase (DBH), and dHAND potentiates the activity of Phox2a. In this study, we use chromatin immunoprecipitation assays to identify target genes of the Phox2 proteins and dHAND. All three proteins are bound to the DBH and PHOX2B promoter regions in SH-SY5Y neuroblastoma cells. The interaction between Phox2a and dHAND is analyzed by fluorescent anisotropy, which demonstrates that dHAND causes an eightfold increase in the affinity of Phox2a for its recognition sites on the DBH promoter region. The Phox2 proteins are not found on the genes encoding other noradrenergic enzymatic or transport proteins but are reciprocally bound to each other’s promoters in SH-SY5Y cells. Together with Phox2a and Phox2b, dHAND is bound to the PHOX2B promoter and is also associated with the GATA2 and eHAND genes in the absence of the Phox2 proteins. These results demonstrate the direct interactions of the Phox2 and dHAND transcription factors within a noradrenergic cell type. The Phox2 proteins were found to share all target genes, whereas dHAND binds to genes independently of Phox2a.
Similar content being viewed by others
References
Adachi M., Browne D., and Lewis E. J. (2000) Paired-like homeodomain proteins Phox2a/Arix and Phox2b/NBPhox have similar genetic organization and independently regulate dopamine beta-hydroxylase gene transcription. DNA Cell Biol. 19, 539–554.
Dai Y. S., Cserjesi P., Markham B. E., and Molkentin J. D. (2002) The transcription factors GATA4 and dHAND physically interact to synergistically activate cardiac gene expression through a p300-dependent mechanism. J. Biol. Chem. 277, 24,390–24,398.
Flora A., Lucchetti H., Benfante R., Goridis C., Clementi F., and Fornasari D. (2001) Sp proteins and Phox2b regulate the expression of the human Phox2a gene. J. Neurosci. 21, 7037–7045.
Goridis C. and Rohrer H. (2002) Specification of catecholaminergic and serotonergic neurons. Nat. Rev. Neurosci. 3, 531–541.
Grillet N., Dubreuil V., Dufour H. D., and Brunet J. F. (2003) Dynamic expression of RGS4 in the developing nervous system and regulation by the neural type-specific transcription factor Phox2b. J. Neurosci. 23, 10613–10621.
Guillemot F, Lo L.-C., Johnson J. E., Auerbach A., Anderson D. J., and Joyner A. L. (1993) Mammalian achaetescute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463–476.
Hong S. J., Kim C. H., and Kim K. S. (2001) Structural and functional characterization of the 5′ upstream promoter of the human Phox2a gene: possible direct transactivation by transcription factor Phox2b. J. Neurochem. 79, 1225–1236.
Howard M., Foster D. N., and Cserjesi P. (1999) Expression of HAND gene products may be sufficient for the differentiation of avian neural crest-derived cells into catecholaminergic neurons in culture. Dev. Biol. 215, 62–77.
Howard M. J., Stanke M., Schneider C., Wu X., and Rohrer H. (2000) The transcription factor dHAND is a downstream effector of BMPs in sympathetic neuron specification. Development 127, 4073–4081.
Huber K., Karch N., Ernsberger U., Goridis C., and Unsicker K. (2005) The role of Phox2B in chromaffin cell development. Dev Biol. 279(2), 501–508.
Huffman J. L., Mokashi A., Bachinger H. P., and Brennan R. G. (2001) The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically. J. Biol. Chem. 276, 40,537–40,544.
Jong Hong S., Chae H., and Kim K. S. (2004) Molecular cloning and characterization of the promoter region of the human Phox2b gene. Brain Res. Mol. Brain Res. 125, 29–39.
Kang S. H., Vieira K., and Bungert J. (2002) Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein-DNA interactions in vivo. Nucleic Acids Res. 30, e44.
Kim C. H., Hwang D. Y., Park J. J., and Kim K. S. (2002) A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene. J. Neurosci. 22, 2579–2589.
Lewis A. L., Xia Y., Datta S. K., McMillin J., and Kellems R. E. (1999) Combinatorial interactions regulate cardiac expression of the murine adenylosuccinate synthetase 1 gene. J. Biol. Chem. 274, 14,188–14,197.
Lim K. C., Lakshmanan G., Crawford S. E., Gu Y., Grosveld F., and Engel J. D. (2000) Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat. Genet. 25, 209–212.
Lo L., Morin X., Brunet J. F., and Anderson D. J. (1999) Specification of neurotransmitter identity by Phox2 proteins in neural crest stem cells. Neuron 22, 693–705.
Lo L.C., Tiveron M. C., and Anderson D. J. (1998) MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity. Development 125, 609–620.
Lundblad J. R., Laurance M., and Goodman R. H. (1996) Fluorescence polarization analysis of protein-DNA and protein-protein interactions. Mol. Endocrinol. 10, 607–612.
Morin X., Cremer H., Hirsch M. R., Kapur R. P., Goridis C., and Brunet J. F. (1997) Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a. Neuron 18(3), 411–423.
Pattyn A., Goridis C., and Brunet J. F. (2002) Specification of the central noradrenergic phenotype by the homeobox gene Phox2b. Mol. Cell. Neurosci. 15(3), 235–243.
Pattyn A., Morin X., Cremer H., Goridis C., and Brunet J. F. (1999) The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399, 366–370.
Rychlik, J. L., Gerbasi V., and Lewis E. J. (2003) The interaction between dHAND and Arix at the dopamine beta-hydroxylase promoter region is independent of direct dHAND binding to DNA. J. Biol. Chem. 278, 49,652–49,660.
Smidt M. P., Smits S. M., and Burbach J. P. (2003) Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur. J. Pharmacol. 480, 75–88.
Stanke M., Junghans D., Geissen M., Goridis C., Ernsberger U., and Rohrer, H. (1999) The Phox2 homeodomain proteins are sufficient to promote the development of sympathetic neurons. Development 126, 4087–4094.
Stanke M., Stubbusch J., and Rohrer H. (2004) Interaction of Mash1 and Phox2b in sympathetic neuron development. Mol. Cell. Neurosci. 25, 374–382.
Swanson D. J., Zellmer E., and Lewis E. J. (1997) The homeodomain protein Arix interacts synergistically with cyclic AMP to regulate expression of neurotransmitter biosynthetic genes. J. Biol. Chem. 272, 27,382–27,392.
Thattaliyath B. D., Firulli B. A., and Firulli A. B. (2002) The basic helix-loop-helix transcription factor HAND2 directly regulates transcription of the atrial naturetic peptide gene. J. Mol. Cell Cardiol. 34, 1335–1344.
Tsarovina K., Pattyn A., Stubbusch J., Muller F., van der Wees J., Schneider C., et al. (2004) Essential role of GATA transcription factors in sympathetic neuron development. Development 131, 4775–4786.
Wells J. and Farnham P. J. (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26(1), 48–56.
Xu H., Firulli A. B., Zhang X., and Howard M. J. (2003) HAND2 synergistically enhances transcription of dopamine-beta-hydroxylase in the presence of Phox2a. Dev. Biol. 262, 183–193.
Yang C., Kim H. S., Seo H., Kim C. H., Brunet J. F., and Kim K. S. (1998) Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine beta-hydroxylase gene. J. Neurochem. 71, 1813–1826.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Rychlik, J.L., Hsieh, M., Eiden, L.E. et al. Phox2 and dHAND transcription factors select shared and unique target genes in the noradrenergic cell type. J Mol Neurosci 27, 281–292 (2005). https://doi.org/10.1385/JMN:27:3:281
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1385/JMN:27:3:281