CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Gene expression regulation in the autonomic nervous system

The high degree of cellular heterogeneity of the mammalian nervous system is due to distinct specification and differentiation processes that mainly rely on transcriptional control mechanisms mediated by transcription factors having discrete temporal patterns of region-specific and cell-type specific expression.

Phox2a and Phox2b are paired-like homeodomain proteins that have been shown to play a pivotal role in the development of the three peripheral divisions of the autonomic nervous system (1). They are also expressed in all of the noradrenergic neurons of the brainstem, in some cranial sensory ganglia that participate in autonomic reflexes, and in a subset of cranial motor neurons (2). None of the components of the autonomic nervous system develop properly in Phox2b knock-out mice (3), whereas Phox2a null mutants show an apparently less severe phenotype that only involves the agenesis of the Locus coeruleus and atrophy of the cranial sensory ganglia (4); nevertheless, they do not feed and die on the day of birth. The different phenotypes of Phox2 knockout mutants along with their asynchronous onset of expression during development underscore that the two factors are not functionally equivalent. This has been more directly demonstrated by reciprocal gene replacement experiments (5) that led to the conclusion that biochemical differences along with different temporal patterns of gene expression may be responsible for the specific function of each paralogue.

One way to understand the specific contribution of each paralogue to the development and functioning of the autonomic nervous system, as the expression of the two factors endures in the adulthood, is to study the genetic mechanisms and the factors involved in their expression. To this aim, the 5'-regulatory region of PHOX2A and PHOX2B human genes have been isolated and their structures and transcriptional activities investigated in cellular models of human autonomic ganglia. Our results showed that PHOX2B binds its own 5'-regulatory region and this auto-regulatory loop is responsible for the 65% of the transcriptional activity of the PHOX2B promoter (6). We also demonstrated that PHOX2B regulates the expression of the PHOX2A gene by directly binding to a site in the region immediately upstream of its minimal promoter (Flora et al, 2001). As preliminary results indicate that PHOX2A can bind the PHOX2B promoter (Cargnign et al, 2001), altogether these data would suggest the existence of a regulatory network, at least in peripheral autonomic ganglia.

Phox2 proteins are also involved in the transcriptional control of the neurotransmitter phenotype (8), as they play a fundamental role in the terminal differentiation of the orthosympathetic system by regulating the gene expression of tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), two limiting enzymes in catecholamine synthesis. Furthermore, we have demonstrated that PHOX2A regulates the expression of alpha3, the ligand-binding subunit of the ganglionic type nicotinic acetylcholine receptor which is a pan-autonomic gene that participates in the fast synaptic transmission between CNS and ganglia (Benfante et al, 2007).

Mutations in human PHOX2A and PHOX2B genes are responsible for genetic diseases,. In particular, mutations in the PHOX2B gene are responsible for the Congenital Central Hypoventilation Syndrome (CCHS), also known as Ondine's curse. Frameshift mutations and polyalanine triplet expansions have been detected in the coding region of PHOX2B in about 90% of CCHS patients, but the molecular pathogenesis of the the disease remains to be elucidate.

 

In order to better understand their function in the development of the entire autonomic nervous system and their pathogenetic role in human genetic diseases it has become essential to identify their target genes. As no information is yet available concerning this fundamental issue, we started to use a genome-wide approach based on chromatin immunopreciptation assay in order to identify the unknown genes directly regulated by PHOX2A and PHOX2B.

References

  1. Howard M. J. (2005) Dev. Biol. 277: 271-86
  2. Brunet J. F. and Pattyn A. (2002) Curr. Opin. Genet. Dev. 12: 435-40
  3. Pattyn A., Morin X., Cremer H., Goridis C. and Brunet, J. (1999) Nature 399: 366-70
  4. Morin X., Cremer H., Hirsch M., Kapu, R. P., Goridis C., and Brunet J. (1997) Neuron 18: 411-23
  5. Coppola E., Pattyn A., Guthrie C. S., Goridis C. and Studer M. (2005) EMBO J. 24: 4392-403
  6. Goridis C. and Rohrer H. (2002) Nat. Rev. Neurosci. 3: 531-41

Publications

  • Benfante R, Antonini RA, Kuster N, Schuderer J, Maercker C, Adlkofer F, Clementi F, Fornasari D (2008) The expression of PHOX2A, PHOX2B and of their target gene dopamine-beta-hydroxylase (DbetaH) is not modified by exposure to extremely-low-frequency electromagnetic field (ELF-EMF) in a human neuronal model. Toxicol In Vitro 22:1489-95.
  • Borghini S, Di Duca M, Santamaria G, Vargiolu M, Bachetti T, Cargnin F, Pini Prato A, De Giorgio R, Lerone M, Stanghellini V, Jasonni V, Fornasari D, Ravazzolo R, Ceccherini I (2007) Transcriptional regulation of TLX2 and impaired intestinal innervation: possible role of the PHOX2A and PHOX2B genes. Eur. J. Hum. Genet. 15:848-55.
  • Benfante R, Flora A, Di Lascio S, Cargnin F, Longhi R, Colombo S, Clementi F, Fornasari D (2007) Transcription factor PHOX2A regulates the human alpha3 nicotinic receptor subunit gene promoter. J. Biol. Chem. 282:13290-302.
  • Cargnin F, Flora A, Di Lascio S, Battaglioli E, Longhi R, Clementi F, Fornasari D (2005) PHOX2B regulates its own expression by a transcriptional auto-regulatory mechanism. J. Biol. Chem. 280:37439-48.
  • Flora A, Lucchetti H, Benfante R, Goridis C, Clementi F, Fornasari D (2001) Sp proteins and Phox2b regulate the expression of the human Phox2a gene. J. Neurosci. 21:7037-45.

Grants

Italian Ministry of Health

Italian Ministry of University and Scientific Research, PRIN

Eagle Foundation, Geneve (Svizzera): EU: SIXTH FRAMEWORK PROGRAMME, LifeSciHealth, Life sciences, genomics and biotechnology for health.

Collaborations

  • C. Goridis, CNRS UMR 8542, Department De Biologie, Ecole Normale Supèrieure, Paris, France.
  • I. Ceccherini, Laboratory of Molecular Genetics, Institute "Giannina Gaslini", Genova, Italy.

 

PI photo

Diego Fornasari

Contact information

email  E-mail

email  +39 02 5031 6960

Participating staff
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