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J. Biol. Chem., Vol. 279, Issue 36, 38062-38071, September 3, 2004

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The Homeoprotein Alx3 Contains Discrete Functional Domains and Exhibits Cell-specific and Selective Monomeric Binding and Transactivation^*

Beatriz Pérez-Villamil||, Mercedes Mirasierra¶, and Mario Vallejo**

From the Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 and Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid, Calle Arturo Duperier 4, 28029 Madrid, Spain

Alx3 is a paired class aristaless-like homeoprotein expressed during embryonic development. Transcriptional transactivation by aristaless-like proteins has been associated with cooperative dimerization upon binding to artificially generated DNA consensus sequences known as P3 sites, but natural target sites in genes regulated by Alx3 are unknown. We report the cloning of a cDNA encoding the rat homolog of Alx3, and we characterize the protein domains that are important for transactivation, dimerization, and binding to DNA. Two proline-rich domains located amino-terminal to the homeodomain (Pro1 and Pro2) are necessary for Alx3-dependent transactivation, whereas another one (Pro3) located in the carboxyl terminus is dispensable but contributes to enhance the magnitude of the response. We confirmed that transcriptional activity of Alx3 from a P3 site correlates with cooperative dimerization upon binding to DNA. However, Alx3 was found to bind selectively to non-P3-related TAAT-containing sites present in the promoter of the somatostatin gene in a specific manner that depends on the nuclear protein environment. Cell-specific transactivation elicited by Alx3 from these sites could not be predicted from in vitro DNA-binding experiments by using recombinant Alx3. In addition, transactivation did not depend on cooperative dimerization upon binding to cognate somatostatin DNA sites. Our data indicate that the paradigm according to which Alx3 must act homodimerically via cooperative binding to P3-like sites is insufficient to explain the mechanism of action of this homeoprotein to regulate transcription of natural target genes. Instead, Alx3 undergoes restrictive or permissive interactions with nuclear proteins that determine its binding to and transactivation from TAAT target sites selected in a cell-specific manner.

Received for publication, January 23, 2004 , and in revised form, June 24, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY488087.

^* This work was supported in part by United States Public Health Service Grant DK-49670 (to M. V.), the Spanish Ministry of Science and Technology Grants PB98-1629-CO2-02 and BMC2002-00870, and the Instituto de Salud Carlos III Grant RGDM G03/212. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Servicio de Oncología Médica, Hospital Clínico San Carlos, 28040 Madrid, Spain.

^¶ Supported in part by a postgraduate fellowship from the Consejo Superior de Investigaciones Científicas.

^** To whom correspondence should be addressed: Instituto de Investigaciones Biomedicas "Alberto Sols," Calle Arturo Duperier, 4, 28029 Madrid, Spain. Tel.: 91-585-4480; Fax: 91-585-4401; E-mail: mvallejo{at}iib.uam.es.

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				M. Mirasierra and M. Vallejo The Homeoprotein Alx3 Expressed in Pancreatic ss-Cells Regulates Insulin Gene Transcription by Interacting with the Basic Helix-Loop-Helix Protein E47 Mol. Endocrinol., November 1, 2006; 20(11): 2876 - 2889. [Abstract] [Full Text] [PDF]