Restriction of Interferon 3' Responsiveness and Basal Expression of the Myeloid Human Fc3'Rlb Gene Is Mediated by a Functional PU.1 Site and a Transcription Initiator Consensus

The restricted expression of the human Fc3,Rlb gene to mydoid cells is likely to be regulated by a combination of transcription factors that may not be solely expressed in myeloid cells, but act together to restrict the expression of the gene to myeloid cells. Low basal expression of the human Fc3,Rlb gene is specifically upregulated by interferon 3' (IFN-3'). A 181-bp region of 5' flanking sequence contains several key regulatory motifs that include the extended gamma response region (XGRR) and the PIE region. The XGRR contains the 39-bp 3" response region originally defined in the highly homologous Fe~Rla gene. The XGRR is in close proximity to the 21-bp PIE motif that is conserved in the promoters of some other myeloid genes. The PIE motif contains a consensus site for the macrophage and B cell transcription factor, PU.1, and is adjacent to the duster of transcription start sites. An active transcription initiator, Iar, consensus spans the start sites and appears to direct transcription initiation of this TATA-less gene. In this study, we demonstrate that the PIE region contains a functional PU.1 site that binds a human PU.l-like protein and that associated factors present in myeloid extracts also bind in this PIE region. Mutational analysis reveals an absolute requirement for an intact PU.1 box for both basal and IFN-3" inducible expression of this gene. In addition, mutations in the Inr greatly reduce basal and inducible transcription. Insertion of a strong TATA box downstream from the Inr or at -30 bp from the transcription start sites restores basal and inducible activity in the presence of a mutated PU.1 site. We also demonstrate that indeed, when the XGRR is positioned in the context of a heterologous TATA containing promoter, it is able to respond equivalently to either IFN-ol or IFN-3". However, IFN-c~ responsiveness does not occur in the context of the physiological Fc3,Rlb TATAlless basal promoter. Our results suggest that a human PU.l-like factor acts as a "bridging factor" between the upstream IFN-3" enhancer and the Inr dependent preinitiation complex. These findings indicate that the structure of the basal promoter in combination with restricted activators like PU.1 are important in regulating the expression of this gene.