A.2 - GENE EXPRESSION REGULATION IN THE AUTONOMIC NERVOUS
SYSTEM
Group Leaders:
Diego Fornasari, Associate Professor of Pharmacology
Roberta Benfante, CNR Researcher
[email protected] / [email protected]
[email protected]
Tel. +39 02 5031 6960 / 6945 – Fax +39 02 74 90 574
http://www.fcm.in.cnr.it/sezione/lines/linea.php?line=2
CURRENT STAFF:
Postdoctoral fellows
Simona Di Lascio
Elena Saba
Undergraduate students
Enrico Cannavò
Fosco Giordano
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
- 11 -
activities investigated in cellular models of human
autonomic ganglia. Our results showed that PHOX2B
binds its own 5’-regulatory region and this autoregulatory 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 panautonomic 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 MJ (2005) Dev Biol 277: 271-86
2. Brunet JF 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, RP, Goridis
C, and Brunet J (1997) Neuron 18: 411-23
5. Coppola E. Pattyn A, Guthrie CS, Goridis C and
Studer M (2005) EMBO J. 24: 4392-403
6. Goridis C and Rohrer H (2002) Nat Rev Neurosci
3: 531-41
Figure 1
PHOX2A and PHOX2B: two transcription factors essentials for the development of the Autonomic Nervous
System
SELECTED PUBBLICATIONS
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-β-hydroxylase (DβH) is not
modified by exposure to extremely-low-frequency
electromagnetic field (ELF-EMF) in a human neuronal
model. Toxicol In Vitro. 22(6): 1489-95.
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 4: 282(18): 13290-302
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(8): 848-55.
- 12 -
Cargnin F, Flora A, Di Lascio S, Battaglioli E, Longhi
R, Clementi F and Fornasari D (2005) PHOX2B
regulates its own expression by a transcriptional autoregulatory mechanism. J Biol Chem 280: 37439-48
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: 7037-45
COLLABORATIONS
C. Goridis, CNRS UMR 8542, Department De
Biologie, Ecole Normale Supérieure, Paris, France
I. Ceccherini, Laboratory of Molecular Genetics,
Institute “Giannina Gaslini”, Genova, Italy
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.