GABP (GA Binding Protein) Is Required for Formation of a Retinoic Acid-Induced Myeloid Enhanceosome on the CD18 Promoter.

CD18, the β chain of the leukocyte integrins, mediates cell-cell and cell-matrix interactions of white blood cells and is required for normal innate immunity. Retinoic acid (RA) induces maturation of myeloid cells; mutations in retinoic acid receptors (RARs) account for acute promyelocytic leukemia. CD18 is transcriptionally regulated by RA in myeloid cells. The proximal promoter of CD18 is transcriptionally regulated by RA but, strikingly, this region is not bound or activated by retinoic acid receptors. Rather, the RA-responsiveness of the CD18 proximal promoter is dependent on ets sites that are bound by the ets transcription factor, GA-binding protein (GABP). The transcriptional co-activator, p300, further increases the RA-responsiveness of CD18. We show that p300 and GABPα, the ets DNA binding subunit of GABP, physically interact in myeloid cells. This interaction uses the cysteine/histidine regions of p300, and the pointed domain (PNT) of GABPα. This demonstrates p300 as the first known interaction partner of the GABPα PNT domain. We prepared a GABPα PNT domain construct for expression in mammalian cells and showed that this PNT construct inhibited the GABPα:p300 interaction and dramatically decreased the RA-responsiveness of the CD18 proximal promoter. We used Chromatin immunoprecipitation (ChIP) to demonstrate that RA induces formation of an enhanceosome on the CD18 promoter; thus, GABPα and p300 on the CD18 proximal promoter recruit RAR/RXR bound to a distal enhancer in the presence of RA. We prepared a dominant negative p300 construct and showed that its expression blocks the formation of the CD18 enhanceosome. These results demonstrate that GABP is required for formation of an enhanceosome that mediates responsiveness of CD18 to RA in myeloid cells. Dominant negative forms of GABPα and p300 interfere with RA responsiveness in myeloid cells and disrupt this enhanceosome. The CD18 enhanceosome is the first known RA-induced enhanceosome and demonstrates its role in mediating retinoid responsiveness in myeloid cells.