Abstract
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.
Highlights
RFX4_v3 is a brain-specific isoform of the transcription factor RFX4
Mouse embryonic stem cells (J1 cells) can differentiate into relatively homogeneous neuronal cells in response to retinoic acid (RA) treatment (22, 23). We found that both Rfx4_v3 and Gps[2] transcripts were up-regulated in J1 cells during neuronal differentiation (Fig. 1C), whereas the Rfx4_v3 message was induced to a much greater extent than the Gps[2] message. These results demonstrate that both Rfx4_v3 and Gps[2] transcripts have similar temporal-spatial distribution patterns within the brain, and they are present in differentiated neuronal cells, suggesting that they could meet under physiological conditions
Since Cx3cl[1] message is present in the testis, our results suggest that RFX4 transcript variant 2 (RFX4_v2) and GPS2 might be involved in the regulation of Cx3cl[1] gene expression in that tissue
Summary
Nuclear Yeast Two-hybrid Screening—Automated yeast twohybrid screening using ProNet technology was performed in conjunction with investigators at Myriad Genetics as previously described with some modifications (12). The cDNA fragments encoding partial human or mouse RFX4_v3 proteins were obtained by PCR amplification and co-transformed with the linear bait vector pGBT.superB into the yeast strain PNY200 (MAT␣ trp[1-901] leu[2,3,112] ura[3-52] his[3-200] ade[2] gal4⌬ gal80) to obtain the bait construct in vivo, in which the partial RFX4_v3 proteins were fused to the carboxyl-terminal end of the Gal[4] DNA binding domain (residues 1–147). The cDNAs from each of three different sources were co-transformed with the prey vector pGAD.PN2 into the yeast strain BK100 (MAT␣ trp[1-901] leu[2,3,112] ura[3-52] his[3-200] gal4⌬ gal[80] LYS2::GAL-HIS3 GAL2-ADE2 met2::GAL7-lacZ) to generate three different prey libraries (human whole brain prey library, ϳ60 million clones; human hippocampus prey library, ϳ38 million clones; mouse embryo prey library, ϳ2.9 million clones), in which prey proteins were fused to the carboxyl-ter-. The partial cDNAs are named according to the corresponding amino acids they encode
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