Regulation Of Transcriptional Inhibition Of RLIM By Rambda And Molecular Mechanism Of RLIM Involved In APL

Tumor suppressor p53 is a key regulator of cell cycle and apoptosis. p53 is a sequence-specific transcription factor that mediates downstream effects by the activation or repression of target genes. The RING finger LIM domain-binding protein RLIM acts as a negative co-regulator for LIM-HD transcription factors via the recruitment of the Sin3A/histone deacetylase corepressor complex. Our previous studies demonstrated that RLIM is a positive regulator of p53 that acts either by inhibiting MDM2-mediated degradation of p53 or by increasing p300-mediated p53 acetylation. However, the mechanism by which RLIM is regulated remains unclear.In this report, we identified RLIM as a novel target of p53 and demonstrated that p53 repressed both mRNA and protein levels of RLIM. Expression of wild-type p53, but not p53 mutants, led to repression of the RLIM promoter. The transcriptional repression of RLIM does not require the direct binding of p53 to the RLIM promoter. Instead, we identified 4 putative Spl binding sites (S1-S4) in the RLIM promoter which are required for p53-mediated repression of RLIM promoter activity. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) indicated that Spl binds to the putative Spl binding sites both in vitro and in vivo. Although p53 does not directly bind to the RLIM promoter, it physically interacts with Spl and inhibits the binding of Spl to the RLIM promoter, which led to the repression of RLIM.We also investigated the role of RLIM in the pathogenesis of acute promyelocytic leukaemia (APL). The promyelocytic leukemia gene (PML) is a tumor suppressor which plays an essential role in apoptosis. PML is typically found concentrated in discrete speckled subnuclear structures which were named PML nuclear bodies (PML-NBs). In APL patients, the t (15; 17) chromosomal translocation happened which fuses the PML gene (on chromosome 15) to the retinoic acid receptor a (RARa) gene (on chromosome 17), leading to the formation of PML-RARa fusion protein. PML-RARa acts as a dominant negative factor of RARa and is responsible for abnormal repression of RARa target genes and the block of hematopoietic differentiation.In the present study, we showed that RLIM interacts with PML both in vitro and in vivo. Overexpression of wild type PML, but not PML-ARING mutant, inhibits RLIM expression. Conversely, konckdown of PML results in stabilization of RLIM. Moreover, the inhibitory role of PML depends on its ability to localize in the PML-NBs. Further studies demonstrated that RLIM interacts with PML through aa 209-430 which contains the nuclear localization sequence (NLS) and the binding domain (BD), and PML interacts with RLIM through the minimal RING domain located in N-terminal. The aa 209-430 region of RLIM which interacts with PML is also essential for the repression and re-localization of RLIM to the PML-NBs by PML. We further found that PML increases RLIM turnover in an ubiquitin-dependent manner and the addition of proteasome inhibitor MG132 abolished the inhibition of RLIM by PML.In APL patients, the t (15; 17) chromosomal translocation generates the PML-RARα fusion protein and disrupts the functions of both PML and RARa. We demonstrated that in APL, RLIM interacts with PML-RARα fusion protein, which results in disruption of the interaction between RLIM and PML. Further studies indicated that RLIM and PML-RARα stabilize each other in vivo. Moreover, RLIM simultaneously interacts with mSin3A and histone deacetylases (HDACs), both of which are the components of N-CoR/SMRT-mSin3A-HDAC co-repressor complex (CoR) recruited by PML-RARα fusion protein in APL. Consequently, RLIM can act as an anchor of PML-RARα and the CoR complex to increase their affinity. Thus, RLIM may participate in the pathogenesis of APL through two ways, to stabilize PML-RARα fusion protein and to enhance the capability of PML-RARα to recruit co-repressor complex.In summary, we identified RLIM as a novel target gene of p53 whose expression is repressed by p53. Meanwhile, RLIM acts as a positive regulator of p53 and enhances its transcriptional activation activities, which forms an autoregulatory feedback loop. These findings shed new light on the relationship between RLIM and p53 and suggested a new pathway by which p53 may affect development. We also defined a novel role of RLIM in the pathogenesis of APL through functional interactions with PML and PML-RARα. This may help us to better understand the molecular mechanisms of leukaemogenesis and provide a potential target for developing therapeutic strategies against APL.