Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains.
Interleukin-5 (IL-5) plays a central role in the differentiation, proliferation, and functional activation of eosinophils. The specific action of IL-5 on eosinophils and hematopoietically related basophils is regulated by the restricted expression of IL-5 receptor alpha (IL-5Ralpha), a subunit of high-affinity IL-5R, on these cells. We have previously identified an enhancer-like cis element in the IL-5Ralpha promoter that is important for both full promoter function and lineage-specific activity. Here, we demonstrate by yeast one-hybrid screening that RFX2 protein specifically binds to this cis element. RFX2 belongs to the RFX DNA-binding protein family, the biological role of which remains obscure. Using an electrophoretic mobility shift assay, we further show that RFX1, RFX2, and RFX3 homodimers and heterodimers specifically bind to the cis element of the IL-5Ralpha promoter. The mRNA expression of RFX1, RFX2, and RFX3 was detected ubiquitously, but in transient-transfection assays, multimerized RFX binding sites in front of a basal promoter efficiently functioned in a tissue- and lineage-specific manner. To further investigate RFX functions on transcription, full-length and deletion mutants of RFX1 were targeted to DNA through fusion to the GAL4 DNA binding domain. Tissue- and lineage-specific transcriptional activation with the full-length RFX1 fusion plasmid on a reporter controlled by GAL4 binding sites was observed. Distinct activation and repression domains within the RFX1 protein were further mapped. Our findings suggest that RFX proteins are transcription factors that contribute to the activity and lineage specificity of the IL-5Ralpha promoter by directly binding to a target cis element and cooperating with other tissue- and lineage-specific cofactors.
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D017930:Genes, Reporter; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D005810:Multigene Family; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000235:genetics*; D053649:Receptors, Interleukin-5; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D012097:Repressor Proteins; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics*