It is still unclear how human cytokine and growth factor receptor genes are regulated during the commitment and differentiation of hematopoietic progenitors to the myeloid lineage in general or to the eosinophil lineage in particular. IL-5 is a late-acting, lineage-specific cytokine which functions on cells of eosinophil lineage (30
). The specific action of IL-5 is mediated through binding to the unique subunit of its receptor, IL-5Rα (41
). Like other hematopoietic growth factor receptors, IL-5Rα plays a critical role in the process of differentiation myeloid progenitors into particular eosinophilic developmental programs. Therefore, it is extremely important to understand the regulation process of IL-5Rα gene expression.
In our previous studies, we identified an enhancer-like element within the functionally active 34-bp region of the IL-5Rα promoter. This element was further narrowed down to a 10-bp region between bp −430 and −421 by methylation interference analysis (Table ) (38
). Based on these results, we inserted the 34-bp element of the IL-5Rα promoter as a bait element in front of a minimum promoter of both HIS
reporter plasmids. We successfully isolated three RFX2 clones from 2 × 106
transformants. Although it has been suggested that at least three tandem copies of the target element are required to activate reporter genes, we have successfully isolated a binding protein by using a single copy of the target element, indicating that the multiple elements may not be critical. Instead, to determine and use a specific target DNA fragment may be a key in yeast one-hybrid screening. It is currently unclear why only RFX2 clones were isolated in our screening, but it seems to be due to the higher number of RFX2 cDNA clones in this particular EML library.
DNA sequence alignment showed that the element from bp −430 to −417 of the IL-5Rα promoter matches the consensus RFX binding site (Fig. A and Table ), which is exactly the same region that we previously identified as a nuclear factor binding site by methylation interference analysis (38
). The formation of two DNA-protein complexes (C1 and C2) was observed in nuclear extracts in our previous experiments. Their identical methylation interference patterns suggested that these complexes were formed by a nuclear factor or factors with the same DNA-binding properties (38
). This previous prediction was confirmed by our present results from supershift experiments. With the specific RFX antibodies, we determined that C1 and C2 complexes represent RFX1 homodimers and RFX1-RFX3 heterodimers, respectively, which is consistent with the result of Northern blot analysis showing predominant expression of RFX1 and RFX3 mRNAs in primary eosinophils. Interestingly, an RFX consensus binding site was also identified in the upstream region of the mouse IL-5Rα promoter by a database search, further indicating the importance of this element.
Although earlier studies have shown that RFX1 is involved in the regulation of the hepatitis B virus enhancer I (33
), no cellular targets for RFX1, RFX2, and RFX3 had been defined. In this report, we have provided several lines of evidence to show IL-5Rα to be the first cellular target regulated by RFX1, RFX2, and RFX3. Point mutation and deletion analyses of the IL-5Rα promoter clearly demonstrated that the nucleotides required for IL-5Rα promoter activity correspond precisely to the nucleotides required for RFX protein binding (Table ). Moreover, transactivation of the IL-5Rα promoter through the RFX element was observed preferentially in myeloid cells. All these findings suggest that RFX proteins play a central role in controlling IL-5Rα gene expression in myeloid cells. To further address the role of RFX in the regulation of IL-5Rα gene expression, we introduced three potential dominant negative mutants of RFX1, which included the DNA binding domain (amino acids [aa] 416 to 620), C terminus (aa 416 to 979), and N terminus (aa 2 to 739), into butyric acid-treated HL-60 7.7 cells by transient transfection. Preliminary experiments showed approximately 1.4- and 1.7-fold declines in the levels of IL-5Rα mRNA in the cells transfected with the C-terminal and N-terminal constructs, respectively (data not shown). This result supports a central role for RFX proteins in the regulation of IL-5Rα expression. However, IL-5Rα mRNA was detected in some, but not all, myeloid lines in which the RFX element is active. This suggests that other transcription factors are also involved in the regulation of IL-5Rα gene expression. In addition to being active in myeloid cells, the enhancer activity of the RFX element was also active in Raji cells (mature B cells), consistent with the fact that the IL-5Rα gene is expressed in B cells in mice (49
Our data and those of others have shown that RFX1, RFX2, and RFX3 are ubiquitously expressed. The RFX proteins were also detected by EMSA in BJA/B, EML, HeLa, and CV-1 cells, in which the RFX element is not transcriptionally active. Moreover, the overexpression of RFX proteins in these cells was not able to induce significant enhancement of transcriptional activity mediated by multimerized RFX elements in vitro or induce expression of the IL-5Rα gene in vivo (data not shown). These results strongly suggest that additional factors are required to cooperate with RFX proteins in controlling IL-5Rα gene expression. With the GAL4-RFX1Full construct, we provided experimental evidence that suggests the involvement of lineage-specific cofactors which modulate RFX function. Several functional domains of RFX1 were mapped by using the GAL4-DBD system (Fig. and ). The amino-terminal domain (residues 1 to 438) functioned as a transactivation domain and activated transcription in all types of cells. A repression domain located around domains B and C showed a common repressive effect on transcription. This domain was able to mask the transactivation mediated by the amino-terminal domain. However, this repression effect could be overcome in a tissue- and lineage-specific manner with full-length RFX1. The carboxy-terminal region (residues 909 to 979) appeared to be important in this process (Fig. B). This region is not able to activate transcription independently; however, it showed a coactivation effect with the amino-terminal domain. It is conceivable that this region interacts with lineage-specific cofactors independently or cooperatively with the activation domain to modulate transcription.
Recent studies of another RFX protein, RFX5, provide a useful model for us to characterize RFX1 function. RFX5 has a DNA binding domain highly characteristic of the RFX family and specifically recognizes a DNA element unique to MHC class II genes (5
). Like other RFX members, RFX5 is ubiquitously expressed. However, it has been demonstrated that transactivation mediated by RFX5 fully relies on a coactivator, CIITA, the expression of which is restricted to dendritic and B cells and is inducible by gamma interferon in a variety of other cell types (17
). RFX5 and CIITA are believed to associate to form a protein complex which is capable of activating transcription from promoters of MHC class II genes (31
). The data in this study suggest that RFX1 may also function through interaction with other lineage-specific cofactors.
The respective roles of RFX1, RFX2, and RFX3 still remain obscure. As shown in Fig. A, they share homologous structures, except for the longer amino terminus of RFX1, and they share the same DNA binding specificity. In addition, the repression domain of RFX1 is localized to the region containing domains B and C, which are highly conserved among these three RFX proteins. These findings are indicative of a redundant function for RFX factors. However, we do not know whether secondary structures formed by each homodimer and heterodimer vary and, therefore, cooperate differently with other proteins. The identification of cofactors associated with RFX1, -2, and -3 proteins is important to further understand the cooperative protein-protein interactions that are required for the transcriptional regulation of the IL-5Rα gene and other myeloid genes as well.