Immunity genes are activated in the Drosophila fat body by Rel and GATA transcription factors. Here, we present evidence that an additional regulatory factor, deformed epidermal autoregulatory factor-1 (DEAF-1), also contributes to the immune response and is specifically important for the induction of two genes encoding antimicrobial peptides, Metchnikowin (Mtk) and Drosomycin (Drs). The systematic mutagenesis of a minimal Mtk 5 enhancer identified a sequence motif essential for both a response to LPS preparations in S2 cells and activation in the larval fat body in response to bacterial infection. Using affinity chromatography coupled to multidimensional protein identification technology (MudPIT), we identified DEAF-1 as a candidate regulator. DEAF-1 activates the expression of Mtk and Drs promoter-luciferase fusion genes in S2 cells. SELEX assays and footprinting data indicate that DEAF-1 binds to and activates Mtk and Drs regulatory DNAs via a TTCGGBT motif. The insertion of this motif into the Diptericin (Dpt) regulatory region confers DEAF-1 responsiveness to this normally DEAF-1-independent enhancer. The coexpression of DEAF-1 with Dorsal, Dif, and Relish results in the synergistic activation of transcription. We propose that DEAF-1 is a regulator of Drosophila immunity.T ranscriptional regulation of Drosophila antimicrobial genes depends on Rel and GATA transcription factors (1-6). Many immunity genes contain tightly linked Rel-and GATAbinding sites in promoter-proximal regions. GATA sites are important for establishing responses in distinct tissues such as the fat body and midgut. Serpent (dGATAb) is thought to regulate antimicrobial gene expression in the fat body (7, 8), whereas dGATAe activates such genes in the midgut in response to ingested microbes (9). In contrast, Dorsal, Dif, and Relish, the NF-B homologues in flies, shuttle between the cytoplasmic and nuclear compartments, acting as ''on/off switches'' for induction (10)(11)(12). Additional factors, such as HOX and POU domain proteins, bind to distal enhancer elements and maintain constitutive domains of gene activity (13). A regulatory element (R1) also has been described within the CecA1 enhancer (14), although the factor that interacts with this motif is unknown.Deformed epidermal autoregulatory factor-1 (DEAF-1) is a transcription factor that was originally shown to bind the autoregulatory enhancer of the Deformed (Dfd) Hox gene, which is activated in embryonic head segments of Drosophila (15). DEAF-1 recognizes several TTCG motifs within the portion of the Dfd autoregulatory region termed ''module E.'' In addition, DEAF-1 binds several similar motifs within a Dfd response element (DRE) from the 1.28 gene that enhances maxillary gene expression during embryogenesis (16). The DEAF-1 binding elements identified in these studies are reportedly not required for enhancer activity however (16,17).The 576-aa DEAF-1 protein possesses two conserved domains, SAND and MYND. The 113-aa SAND domain (named for SP100, AIRE-1, NucP41/75, and DEAF-1) (18) i...
Triple-negative breast cancer [TNBC, lacks expression of estrogen receptor (ER), progesterone receptor (PR) and amplification of HER2/Neu] remains one of the most aggressive subtypes, affects the youngest patients and still lacks an effective targeted therapy(1,2). Both phosphatidylinositol-3-kinase (PI3K)-α and -β contribute to oncogenesis of solid tumors, including the development of breast cancer(3). Inositol polyphosphate-4-phosphatase type II (INPP4B) catalyzes the removal of the 4′-phosphate of phosphatidylinositol-(3,4)-bisphosphate (PI-3,4-P2) creating phosphatidylinositol-3-phosphate(4). There is debate concerning whether PI-3,4-P2 contributes to Akt and downstream effector activation with the known canonical signaling second messenger, phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) (5–7). If PI-3,4-P2 is a positive effector, INPP4B would be a negative regulator of PI3K signaling and there is some evidence to support this(4,8). Utilizing phosphatase and tensin homolog deleted on chromosome ten (PTEN)-null triple-negative breast tumor cell lines, it was unexpectedly found that silencing INPP4B decreased basal phospho-Akt (pAkt) and cellular proliferation, and in most cases sensitized cells to PI3K-α and -β isoform-specific inhibitors. Conversely, overexpression of INPP4B desensitized cells to PI3K inhibitors in a phosphatase activity-dependent manner. In summary, the current investigation of INPP4B in PTEN-null TNBC suggests new mechanistic insight and the potential for targeted therapy for this aggressive subset of breast cancer. Implications These data support a model where PI-3,4-P2 is inhibitory toward PI3K, revealing a novel feedback mechanism under conditions of excessive signaling, and potentially an indication for PI3K-β isoform-specific inhibitors in PTEN-null TNBC that have lost INPP4B expression.
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