In this report, DNase I hypersensitivity implicated untranslated exon 1 in regulating CCR3 transcription. Furthermore, nuclear proteins derived from eosinophilic cells were shown to bind CCR3 exon between nucleotides +25 to +60. Using unlabelled competitors and antibodies, proteins of the GATA family, specifically GATA-1, were shown to bind to this region. Taken together, these data suggest that untranslated exon 1, via GATA-1, has a regulatory role in CCR3 transcription. Finally, we demonstrate that the 1.6-kb CCR3 promoter element, that includes exon 1, is broadly active in vivo.
The HS1 site was located in the vicinity of exon 1. Combined with our previous results demonstrating diminished promoter activity when exon 1 is deleted from the promoter construct, these data suggested that 5' untranslated exon 1 may have a regulatory function. It has been reported that the 5' untranslated exons may contain sequences that facilitate transcription of the gene. Examples include a GATA-1 site in the 5'-UTR of the γ-globin gene, an HNF-1 site in the plasminogen gene, a PU.1 site in the PU.1 gene and a C/EBP site in the CCR2 gene [24
]. Thus, we hypothesized that nuclear proteins bind to exon 1. Our EMSA analysis, coupled with cold competitors and specific antibodies, indicates that proteins of the GATA family, specifically GATA-1 bind to the CCR3 exon 1. Thus, GATA-1 binding to exon 1 may regulate CCR3 transcription. Alternatively, GATA-1 binding to exon 1 may affect transcription start site function, RNA stability or translation. These possibilities will be addressed in future studies.
It is important that this data be viewed with what is known about other myeloid-specific promoters, that have often proven to be difficult to function independently in vivo
. For example, constructs using the 5' flanking region of myeloid-specific genes have not been useful for transgenic work (such as the CD14 promoter [36
], the c-kit promoter [37
], or the 1.7 kb CD11b promoter [38
]). Better success was obtained when the entire gene, including the open reading frame, was used (e.g. the human cathepsin G, chicken lysozyme and c-fps/fes transgenic constructs [39
]). These constructs were at least 6 kb in size and contained all exons and introns and several kb of 5' and 3' flanking sequence. Presumably, these larger constructs contained the locus control region (LCR)- sequences that have the ability to dominantly control gene expression in any chromosomal region. This in turn allows for a high degree of consistency among independent mouse lines with regard to cell specificity, level of expression and proportionality to gene copy number. These regions may be located at several different sites in the gene, including introns and coding exons; thus, screening with DNase I hypersensitivity is usually the first method employed to identify these regions. Transgenic mice expressing the EGFP reporter gene under the control of the CCR3 promoter demonstrate that the 1.6 kb promoter and 60 bp of exon 1 of the CCR3 gene confer strong promoter activity in vivo
. However, these sequences do not contain the entire LCR, since expression of the reporter gene was variable among multiple founder lines. DNase I hypersensitivity studies discovered one hypersensitive site in the CCR3 locus. The HS1 site was apparently not sufficient for integration-site independent effects, since it is contained in the promoter construct used. Thus, future studies will need to broaden the search for the CCR3 LCR. It is important to note that the true cellular specificity of CCR3 has not been established. While this gene product is often considered to be specific for inflammatory cells involved in allergic inflammation (eosinophils, mast cells, and possibly Th2 cells), several reports have documented expression by other cell types including additional leukocytes (e.g. dendritic cells), as well as tissue cells (epithelial and endothelial cells) [11
]. Thus, it remains to be determined if our observation that the CCR3 promoter has broad activity in vivo
represents the true endogenous activity.
Since CCR3 is expressed strongly on eosinophils, analysis of the signals that induce its expression may give insight into the molecular mechanisms for the commitment of myeloid progenitors to the eosinophil lineage. It is generally believed that transcription factors are the final common pathway driving differentiation and that hematopoietic commitment to different lineages is driven by alternative expression of specific combinations of transcription factors [43
]. Although no eosinophil-specific transcription factors have been reported, eosinophil commitment appears to be regulated by GATA-1, PU-1 and C/EBP proteins [34
]. Consistent with this, DNA binding sites for these transcription factors are found in several eosinophil-selective promoters, such as the promoter for major basic protein (MBP), IL-5 receptor alpha (IL-5Rα) chain and Charcot-Leyden crystal (CLC) protein. Specifically, ectopic overexpression of GATA-1 in chicken myeloblasts leads to transdifferentiation into eosinophils or thromboblasts depending on the dose used [47
]. GATA-1 transactivates several eosinophil-selective promoters [48
] including the avian EOS47 promoter in which the GATA-1 site is located downstream of the transcription start site [34