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Caterina Missero, Ph.D. Group
Leader,
Centre of Genetics Engineering E-mail: missero@ceinge.unina.it Ph.D. in Biology, University of Trieste (Trieste, Italy), 1989 |
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Molecular
basis of skin development and disease. Our
research is focused on studying
transcriptional mechanisms and genetic pathways required for normal skin
development, and on the identification of genetic alterations that occur in
inherited and in acquired skin diseases. Stratified epithelia of the
skin, such as the epidermis and the hair follicle, are constantly self-renewing
tissues that provide a fascinating system to study the molecular and cellular
mechanisms governing tissue formation and homeostasis. Using primary keratinocytes derived from human and mouse skin, as well
as mouse genetic models, we are investigating how epithelial cells in the
skin initiate stratification and regulate their differentiation to establish
unique programs of gene expression. To
tackle this problem we are taking functional genomics and molecular biology
approaches starting from the identification of upstream regulators and
downstream effectors of the transcription factor p63. Its homozygous deletion
in mice results in the absence of all stratified epithelia and their derivatives,
while deregulated expression of p63
has been observed in squamous cell carcinomas. Our work reveals that p63 promotes cell proliferation, whereas it
suppresses terminal differentiation. In addition we unveil a crucial role of
p63 in maintaining epidermal cell identity. Pathways
downstream of p63. p63
is involved in many cellular processes. We recently identified a number of
p63 target genes using genome-wide expression profiling and Chromatin immunoprecipitation on a chip (ChIP-chip) (Della Gatta
et al., 2008). To identify immediate
early-responsive genes, we engineered a tamoxifen-responsive
p63 protein (ERp63). Direct and functional targets can be transiently
activated by p63, and experiments that do not take into account temporal
dynamics may fail to identify such targets. Using
our approach, we uncovered a previously unsuspected transient regulation of
the AP-1 complex by p63 through direct regulation of a subset of AP-1
components. In addition we found a large number of genes directly regulated by
p63. p63 induces several genes
involved in cell cycle progression, thus positively controlling
proliferation. In contrast, p63 also
directly suppresses a subset of genes encoding for late differentiation
markers, while affecting others through suppression of Notch1 signaling. In keratinocytes Notch1 is required to restrict growth and promote
differentiation, whereas p63 plays the opposite function. We demonstrated
that p63 negatively modulates Notch1 function by directly suppressing the
expression of the Notch1 downstream effector Hes-1 (Nguyen et al., 2006).
Conversely, p63 expression is suppressed by Notch1 activation through a mechanism
requiring down-modulation of selected interferon-responsive genes. Thus, a
complex cross-talk between Notch and p63 is involved in the balance between keratinocyte self-renewal and differentiation. In a parallel approach, we identified a novel skin-specific gene Tprg (Transformation related
protein 63 regulated) that is directly
regulated by p63 through a long-distance enhancer (Antonini
et al., 2008). Tprg
is located upstream of the p63 gene
in the vertebrate genome. p63 and p73 are
highly homologous members of the p53
family that originated by gene duplication at the invertebrate-to-vertebrate
transition. We found that Tprg has striking similarity to an uncharacterized gene
located upstream of p73, which we
named Tprgl (Tprg-like),
thus demonstrating that p63/Tprg and p73/Tprgl are embedded
in a previously unidentified paralogue region
originated from a single duplication event. Tissue-specific regulation of p63 expression. p63 is one
of the earliest markers of stratified epithelia during development, however
the mechanisms controlling p63 expression are still poorly understood. Using
a genomic sequence comparison approach across multiple vertebrate species, we
isolated a highly conserved distal enhancer in the p63 locus that confers
strong tissue-specific activity in transgenic mice (Antonini
et al., 2006). Functional characterization of the enhancer has revealed an autoregulatory feedback loop in which the p63 protein
directly binds and is an essential regulator of the enhancer. We are
currently searching for other genomic elements in the p63 locus that
contribute to regulate p63 gene expression. These studies will be crucial to
identify major determinants of gene expression in stratified epithelia. The Shh pathway The Sonic hedgehog (Shh)
pathway plays a critical role in hair follicle physiology, and is
constitutively active in several human tumors including basal cell carcinomas
(BCC), the most common human malignancy. We demonstrated that Shh signaling positively regulates the expression of the Foxe1 gene, encoding a transcription
factor required for proper hair follicle morphogenesis (Brancaccio et al.
2004). Consistently, Foxe1 is
aberrantly expressed in BCC, while it is undetectable in normal epidermis and
squamous cell carcinoma. To establish the role of
Foxe1 in BCC we are generating mouse models lacking Foxe1 in skin and
crossing them with a genetic model of BCC development. We also demonstrated that Shh and its effector Gli2
directly induce the thyroid hormone-inactivating enzyme Dio3 (Type 3 iodothyronine deiodinase) in proliferating keratinocytes
and in mouse and human BCCs (Dentice
et al., 2007). Dio3 induction reduces intracellular active thyroid hormone,
thus resulting in increased keratinocyte
proliferation. Importantly, Dio3 knockdown caused a significant reduction in
BCC development in a xenograft model. This
previously unrecognized functional cross-talk between Shh/Gli2 and the
thyroid hormone and offers a potential therapeutic approach to BCC (see
patent).
Figure 1. Identification
of p63 target genes. (A) p63 was fused to a
modified estrogen receptor domain (ERp63) and expressed in primary mouse keratinocytes. Upon treatment with estrogen agonist tamoxifen, total RNA was collected at 10-min intervals
for the first hour, and then at 20-min intervals until 4 h. Expression
profiles of p63-responsive genes following p63 activation is shown as log2 ratio of mRNA
levels versus an untreated control. (B) Upregulated
(left panel) or downregulated (right panel)
transcripts were classified according to their biological categories as
indicated in the pie charts. Bibliography Antonini, D., Rossi, B., Han, R., Minichiello, A., Di Palma, T.,
Corrado, M., Banfi, S., Zannini, M., Brissette, J.L., Missero, C. (2006). An
evolutionarily conserved long-range enhancer controls p63 expression through
a positive autoregulatory loop. Mol. Cell. Biol., 2006;26 3308-3318. Antonini,
D., Dentice, M., Mahtani, P., De Rosa, L., Della Gatta, G., Mandinova, A.,
Salvatore, D., Stupka, E., Missero, C. (2008). Tprg, a gene
predominantly expressed in skin, is a direct target of the transcription
factor p63. J Invest Dermat, 128(7):
1676-1685. Brancaccio, A., Minichiello, A., Grachtchouk, M.,
Antonini, D., Sheng, H., Parlato, R., Dathan, N., Andrzej A.
Dlugosz, Missero, C. (2004). Requirement
of the forkhead gene Foxe1, a target of sonic hedgehog signaling, in hair follicle
morphogenesis. Hum.
Mol. Gen., 13 (21): 2595-2606. Della Gatta, G., Bansal, M.,
Ambesi-Impiombato, A., Antonini, D.,
Missero*, C., di Bernardo*, D. (2008). Direct targets of the Trp63
transcription factor revealed by a combination of gene expression profiling
and reverse engineering. Genome Research, 18(6): 939-48. (*co-corresponding
author and equal contribution). Dentice, M., Luongo,
C., Huang, S., Ambrosio, R., Elefante,
A., Mirebeau-Prunier, D., Zavacki,
A.M., Fenzi, G., Grachtchouk,
M., Hutchin, M., Dlugosz,
A.A., Bianco, A.C., Missero,
C., Larsen, P.R., Salvatore, D. (2007). Sonic hedgehog-induced type 3 deiodinase blocks thyroid hormone action enhancing
proliferation of normal and malignant keratinocytes.
Proc Natl Acad Sci U S A,104(36):14466-71. Nguyen, B.-C., Lefort, K., Mandinova, A., Antonini, D., Devgan, V., Della Gatta, G., Koster, M.I., Zhang, Z., Wang, J., Tommasi di Vignano, A., Kitajewski, J., Chiorino, G., Roop, D.R., Missero*, C., Dotto*, G.P. (2006). Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation. Genes Dev., 2006; 20 1028-1042. (*co-corresponding author and equal contribution). |
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