Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.

Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.

HIV-1 Nef mediates post-translational down-regulation and redistribution of the mannose receptor.

Human immunodeficiency virus (HIV) has derived a variety of means to evade the host immune response. HIV-derived proteins, including Tat, Nef, and Env, have all been reported to decrease expression of host molecules such as CD4 and major histocompatibility complex I, which would assist in limiting viral replication. The mannose receptor (MR) on the surface of macrophages and dendritic cells (DC) has been proposed to function as an effective antigen-capture molecule, as well as a receptor for entering pathogens such as Mycobacterium tuberculosis and Pneumocystis carinii. Regulation of this receptor would therefore benefit HIV in removing an additional arm of the innate immune system. Previous work has shown that MR function is reduced in alveolar macrophages from HIV-infected patients and that surface MR levels are decreased by the HIV-derived protein Nef in DC. In addition, several laboratories have shown that CD4 is removed from the surface of T cells in a manner that might be applicable to decreased MR surface expression in macrophages. In the current study, we have investigated the role of Nef in removing MR from the cell surface. We have used a human macrophage cell line stably expressing the MR as well as human epithelial cells transiently expressing CD4 and a unique CD4/MR chimeric molecule constructed from the extracellular and transmembrane domains of CD4 and the cytoplasmic tail portion of the MR. We show that the MR is reduced on the cell surface by approximately 50% in the presence of Nef and that the MR cytoplasmic tail can confer susceptibility to Nef in the CD4/MR chimera. These data suggest that the MR is a potential intracellular target of Nef and that this regulation may represent a mechanism to further cripple the host innate immune system.

IL-4 modulates transcriptional control of the mannose receptor in mouse FSDC dendritic cells.

The mannose receptor is a 175 kDa protein found on the surface of macrophages and dendritic cells whose functions include clearance of extracellular hydrolases, internalization of pathogens, and antigen capture. Receptor expression is closely linked to the functional state of these cells and is regulated by cytokines. Previous work has shown that treatment of macrophages and dendritic cells with interleukin-4 leads to increased mannose receptor expression. We have examined the mechanism of this IL-4-mediated up-regulation in the murine dendritic cell line FSDC. IL-4 increased mannose receptor activity, protein, and mRNA. The mannose receptor promoter was functional in FSDCs using transient transfection assays, and IL-4 treatment increased promoter activity 2.6-fold. The responsive region was localized to the proximal 228 bp. Electrophoretic mobility shift assays detected an IL-4-inducible protein that bound to the mannose receptor promoter at a site spanning the region between -147 and -108 bp. The sequence TTAC(N)4CACC (-135 and -124 bp) is similar to the IL-4 response region in the Fc receptor II. Mutation of the flanking TT and CC in this motif blocked IL-4 responsiveness and binding of the IL-4-induced mannose receptor binding protein. This protein does not appear to be STAT6 since neither an anti-STAT6 antibody nor a STAT6 consensus oligonucleotide altered factor binding.