PC2, the high-molecular weight constituent of the potent USA transcriptional coactivator fraction, was identified as a Mediator-like complex. Its composition resembles that of the TRAP/SMCC complex, but PC2 is distinguished by the prominent absence of the SRB10 and SRB11 kinase/cyclin pair, as well as several additional polypeptides. Furthermore, affinity-purified PC2, which lacks independent activity, acts in synergy with USA-derived coactivators PC3/topoisomerase I and PC4 to mediate the effects of a variety of activators (including VP16, the synthetic activator GAL4-AH, and the orphan nuclear receptor HNF4) and thus recapitulates partial USA coactivator function.

The TRAP/Mediator complex serves as a coactivator for many transcriptional activators, including nuclear receptors such as the thyroid hormone receptor (TR) that targets the TRAP220 subunit. The critical but selective function of TRAP220 is evidenced by the embryonic lethal phenotype of Trap220 ؊/؊ mice and by the observation that Trap220 ؊/؊ fibroblasts (isolated before embryonic death) are impaired in specific nuclear receptor-dependent pathways. Here we have used a biochemical and genetic approach to understand the basis of specificity in TRAP220 function. We show that Trap220 ؊/؊ cells possess a TRAP/Mediator complex that is relatively intact and compromised in its ability to support TR-dependent, but not VP16-dependent, transcription in vitro. Transfection studies using TRAP220 mutants revealed that the N terminus of TRAP220 is necessary and sufficient for stable association with the TRAP/Mediator complex and, further, that TRAP220-dependent TR function in transfected cells requires both of the NR boxes that contain the LXXLL motif implicated in nuclear receptor binding. Similarly, an analysis of isolated TRAP/Mediator complexes with mutations in either or both of the two NR boxes confirmed a critical role for them in in vitro coactivator function. The implications of these observations are discussed in terms of our present understanding of coactivator function.Nuclear receptors constitute a large superfamily of transcription factors that control diverse biological processes such as cell growth, differentiation, and homeostasis (reviewed in references 30 and 45). In the vast majority of cases, nuclear receptor functions are accomplished through a series of molecular events that are triggered by the binding of the cognate ligand. The liganded nuclear receptor, which binds to regulatory elements of target genes, orchestrates the assembly of multiprotein complexes containing various coactivators that directly or indirectly lead to enhanced activity of the preinitiation complex (PIC), which consists of RNA polymerase II (Pol II) and its associated basal transcriptional machinery (reviewed in references 37 and 38).The various coactivators that have been implicated in the function of nuclear receptors and other transcriptional activators fall into two broad categories. One group is comprised primarily of cofactors that facilitate the initial penetration of chromatin (10, 13, 50) either through ATP-dependent remodeling (SWI/SNF and various I-SWI-containing complexes) or through covalent protein modifications such as histone acetylation (p160 family members, CBP/p300, GCN5-containing SAGA complex) or methylation (CARM1 and PRMT) (41). The other group contains coactivators that were identified, for the most part, by biochemical assays utilizing naked DNA templates and are thus thought to function directly at the level of PIC formation or function (37). This group includes the TATA box binding protein (TBP)-associated factors in TFIID (46); positive cofactors PC1, PC2, PC3, PC4, and PC52 (37) derived from the USA fra...

The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma in the adult population, and treatment of DLBCL is still unfavorable. Therefore, there is an urgent requirement to investigate the molecular mechanisms underlying DLBCL tumorigenesis. To study the potential function of microRNA-155 (miR-155) involved in the regulation of lymphoma, we monitored lymphoma cell behavior including proliferation, cell cycle, and apoptosis using CCK-8 and flow cytometry analysis. Real-time PCR was used to detect the expression levels of miR-155 in 118 lymphoma patients' tissues, and Western blot was also used to analyze the expression level of proteins correlated with cell cycle and apoptosis in lymphoma cells. miR-155 expression levels were higher in lymphoma tissues compared with adjacent tissues. Downregulation of miR-155 inhibited lymphoma cell progress by arresting cell cycle in the G0/G1 phase and promoting apoptosis. Cell cycle-correlated proteins (cyclin B1, cyclin D1, and CDK4) were inhibited by downregulation of miR-155. Apoptosis-correlated proteins level (Bax/Bcl-2 and caspase 3 activity) were increased by downregulation of miR-155. In addition, a significant inverse correlation between the level of miR-155 and transforming growth factor-β receptor 2 (TGFBR2) was observed, which has been demonstrated to be a novel tumor suppressor gene. A further in vivo tumor formation study in nude mice indicated that downregulation of miR-155 in lymphoma cells delayed the progress of tumor formation. These findings indicate that miR-155 may serve as a useful potential target for the treatment of lymphoma.

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