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J. Biol. Chem., Vol. 283, Issue 13, 8580-8590, March 28, 2008

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G-protein Pathway Suppressor 2 (GPS2) Interacts with the Regulatory Factor X4 Variant 3 (RFX4_v3) and Functions as a Transcriptional Co-activator^*

Donghui Zhang, G. Jean Harry, Perry J. Blackshear^¶||, and Darryl C. Zeldin^¶1

From the Laboratories of Respiratory Biology and Neurobiology and the ^¶Office of Clinical Research, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ^||Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

RFX4_v3 (regulatory factor X4 variant 3) is a brain-specific isoform of the transcription factor RFX4. Insertional mutagenesis in mice demonstrates that Rfx4_v3 is crucial for normal brain development. Many genes involved in critical processes during brain morphogenesis are dysregulated in Rfx4_v3 mutant brains. For example, Cx3cl1 is a CX3C-type chemokine that is abundant in brain and is a direct transcriptional target of RFX4_v3 through a specific promoter X-box (X-box 1), the responsive element for RFX proteins. To identify potential interacting partners for RFX4_v3, we performed yeast two-hybrid analysis. Nine candidate interactors were identified, including GPS2 (G-protein pathway suppressor 2). Indirect immunofluorescence demonstrated that GPS2 and RFX4_v3 co-localized to the nucleus. Both GPS2 and RFX4_v3 mRNAs were also present in most portions of the adult mouse brain as well as in brains at different ages, suggesting that the two proteins could bind to each other. Co-immunoprecipitation assays indicated that physical interactions between GPS2 and RFX4_v3 did indeed occur. Furthermore, GPS2 was recruited to the Cx3cl1 promoter by RFX4_v3 and potentiated RFX4_v3 transactivation on this promoter through X-box 1, suggesting that the protein-protein interaction was functionally relevant. GPS2 bound to both the carboxyl-terminal region (amino acids 575-735) and the middle region (amino acids 250-574) of the RFX4_v3 protein. RFX4_v3 amino acids 1-574 stimulated the Cx3cl1 promoter to a similar extent as the full-length RFX4_v3 protein; however, deletion of the carboxyl-terminal region of RFX4_v3 impaired the co-activating abilities of GPS2. Based on these data, we conclude that GPS2 interacts with RFX4_v3 to modulate transactivation of genes involved in brain morphogenesis, including Cx3Cl1.

Received for publication, October 3, 2007 , and in revised form, January 22, 2008.

^* This research was supported by the Intramural Research Program of NIEHS, National Institutes of Health. 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.

¹ To whom correspondence should be addressed: National Institutes of Health/NIEHS, 111 T.W. Alexander Dr., Bldg. 101, Rm. D236, Research Triangle Park, NC 27709. Tel.: 919-541-1169; Fax: 919-541-4133; E-mail: zeldin{at}niehs.nih.gov.

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