Abstract 250The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme. Mediator acts as the end-point integrator of a variety of activators and intracellular signaling, and conveys these signals to the general transcription machinery. Among circa 25 subunits, MED1/TRAP220 subunit is crucial for many biological events through the interaction with distinct activators such as nuclear receptors (NRs) and GATA family activators. In hematopoiesis, MED1 plays an important role for optimal NR-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we analyzed the role of MED1 in the niche. We used mouse embryonic fibroblasts (MEFs) as an in vitro niche model, since MEFs have a nuance of the osteoblastic precursor and support long-term culture-initiating cells (LTC-ICs). When syngenic normal bone marrow (BM) cells were cocultured with mitomycin C-treated Med1−/− or Med1+/+ MEFs (in p53−/− background), the number of live cells (cell counts, DNA contents and MTT assays) and DNA synthesis (BrdU incorporation) was significantly suppressed on Med1−/− MEFs compared with the control during the two-week period. However, they recovered to the control level when cocultured on Med1−/− MEFs into which MED1 was stably introduced (Rev-Med1−/− MEFs). Furthermore, when LTC-ICs were assayed in complete methylcellulose media (Methocult M3434, StemCell Technologies) after the 8-week coculture, the number of LTC-ICs was attenuated for BM cells cocultured on Med1−/− MEFs compared to the control, but recovered to the control level when cocultured on Rev-Med1−/− MEFs. In order to identify the direct target(s) of MED1 that was responsible for these phenotypes, a microarray analysis of mRNA comparing Med1+/+ and Med1−/− MEFs was performed, and disclosed approximately 15 genes whose expressions were profoundly attenuated in Med1−/− MEFs. Among molecules encoded by these genes we focused on osteopontin (OPN) because solely OPN was known to have a role in niche to support hematopoietic stem/precursor cells (HSPCs). Angiopoietin-1 and Jagged-1 expressions were comparable. The expression of Opn mRNA was distinctly downregulated in Med1−/− MEFs but recovered to the control level in Rev-Med1−/− MEFs. The Opn expression in Med1+/+ MEFs was also similar to those in MC3T3-E1 mouse osteoblastic, and OP-9 BM stromal, cells. Western blot analysis of Med1+/+ MEFs using the polyclonal antibody that recognized the N-terminus of mouse OPN disclosed the abundant expression of the full-length form of OPN (ca. 70kDa), which was much less in Med1−/− MEFs. In contrast, the N-terminal cleaved form of OPN was barely visible in both MEFs. Next a chromatin immunoprecipitation (ChIP) assay was performed to know if Mediator complex was actually recruited to the Opn promoter. As expected, when the sheared chromatin was immunopurified with the antibody against MED10 subunit, Mediator was proved to be recruited to the Opn promoter more abundantly in Med1+/+ than Med1−/− MEFs, and the recruitment in Rev−Med1−/− MEFs recovered to the level of the former. Transient transfection and luciferase reporter assays disclosed that the basal level transcription as well as vitamin D receptor (VDR)- and Runx2-mediated transcriptional activation of Opn was specifically attenuated in Med1−/− MEFs, and that the basal transcription and ligand-dependent activation were dependent on the N-terminal domain (amino acids 1 to 602) and the two NR-recognition motifs of MED1, respectively. If OPN was the direct target of MED1, OPN might be responsible for, and the addition or depletion of OPN might alter, the BM cell growth and LTC-ICs support. Indeed, the addition of recombinant full-length OPN to Med1−/− MEFs restored, and the addition of the anti-OPN (N-terminus) polyclonal antibody to Med1+/+ MEFs attenuated, both the growth of cocultured BM cells during the two-week coculture and the numbers of LTC-ICs after the long-term coculture. Finally, to exclude the possibility that these phenotypes might have been restricted to MEFs, OP-9 cells, which had widely-accepted niche function, were used for similar experiments. Indeed, the addition of the anti-OPN antibody to OP-9 cells attenuated both the growth of cocultured BM cells and the numbers of LTC-ICs. Taken together, these data suggest that MED1 in niche cells, through upregulating VDR- and Runx2-mediated transcription on the Opn promoter, plays an important role in HSPCs support. Disclosures:No relevant conflicts of interest to declare.