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
Postdoctoral fellow, Department of Pathology, School of Medicine, Yale University (New Haven, USA), 1989-1992
Research Scientist, Cutaneous Biology Research Center, Massachusetts General Hospital (Boston, USA), 1992-1996
Instructor in Dermatology, Harvard Medical School (Boston, USA), 1992-1996
Research Scientist, Biochemistry and Molecular Biology Department, Stazione Zoologica “A. Dohrn” (Napoli, Italy), 1996-2000
Group Leader, Telethon Institute of Genetics and Medicine (TIGEM, Napoli, Italy) 2000-2006 Group
Group Leader, CEINGE Biotecnologie Avanzate  (Center for Genetic Engineering, Napoli, Italy) 2006-to-date

 

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).