The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1–602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1–602) into Med1−/− mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1–602)-interacting coactivator CCAR1, CoCoA, and MED1(681–715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA, and MED1(1–602) formed a complex in vitro, and GATA1, CCAR1, CoCoA, and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.
The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme and acts as the end-point integrator of a variety of activators and intracellular signalings. The Mediator activates transcription in concert with a group of recruited general transcription initiation factors. Among the circa 25 subunits, the MED1/TRAP220 subunit was originally demonstrated by us as a key component that acts as a ligand-dependent nuclear receptor-specific coactivator, and proved to play an important role in retinoic acid-dependent granulocytic and 1,25-dihydroxyvitamin D3-dependent monocytic differentiation of both normal hematopoietic precursor and acute promyelocytic leukemia cells, via retinoic acid receptor (RAR) and vitamin D receptor (VDR), respectively. MED1 was recently re-identified as a specific coactivator for GATA family activators and found to function in the process of GATA-1-mediated normal erythroblast differentiation. In this study, we analyzed the role of MED1 in erythroid differentiation of malignant erythroblastic (leukemic) cells. We used K562 human erythroleukemia cells as a model since GATA-1-mediated erythroid differentiation of K562 is efficiently induced with hemin. When K562 cells were exposed to 50nM hemin for 5 days, 44% of cells became positive for benzidine staining and the expression of the erythroid lineage-specific (and GATA-1-targeted) genes such as β-globin, γ-globin, porphobilinogen deaminase (PBGD) and 5-aminolevulinate synthase (ALAS-E) were strongly induced. The Mediator subunits including MED1 were also induced strongly together with these erythroid-specific genes in this process. Since the precedent study has demonstrated that Mediator subunits are induced during nuclear receptor-mediated myelomonopoiesis, and since genes whose products have physiological significance are often induced, Mediator might be employed in GATA-1-mediated erythroid differentiation of erythroleukemia cells as well. Then we focused on the GATA-1-specific coactivator MED1 among the Mediator subunits. In order to analyze the role of MED1, we first reduced the expression of endogenous MED1 in K562 cells with the MED1 knockdown small hairpin RNA (shRNA) expression vector. When the transiently knocked down cells (approximately 25% of the control) were treated with hemin, the number of benzidine-positive cells were half of the control in 3 days, indicating that hemin-induced erythroid differentiation was strongly inhibited. Further, the expression of direct target genes for GATA-1 such as β-globin, γ-globin, PBGD and ALAS-E were significantly reduced by approximately 70% in knockdown cells. Moreover, when the knockdown cells were cultured for 3 days without hemin, the basal expression levels of the erythroid lineage-specific genes such as β-globin, γ-globin and PBGD were significantly reduced by approximately 30%. We next asked if overexpressed MED1 might enhance erythroid differentiation. Since the MED1 domain near the N-terminus (a.a. 622 to 701) interacts with GATA-1 and that the N-terminal domain that contains two LxxLL nuclear receptor recognition motifs (NR box; aa. 592 to 703) is sufficient for in vitro and most in vivo transcription events, we tested the N-terminal truncation of MED1, N-MED1 (aa. 1 to 703), for overexpression. When N-MED1 was stably expressed in K562 cells after selection with G418, MED1 expression was boosted at least 2.2 folds of the control transfectant cells. When these cells were treated with hemin, benzidine-positive differentiated cells were 3-fold reduced and the expression of the erythroid differentiation marker genes (above) were approximately 2-fold increased in N-MED1 cells after one day. However, after 3 days, the number of benzidine-positive cells and the expression of erythroid marker genes in N-MED1 cells were saturated and caught up by the control cells. Thus, it appears that overexpressed MED1 enhances the erythroid differentiation in K562 cells. Together with the finding that GATA-1 is physically associated with MED1 and that MED1 is co-occupied with GATA-1 in the enhancer region, these results indicate that MED1 functions as a GATA-1-specific coactivator in erythroid differentiation of not only normal erythroblasts but erythroleukemic cells. MED1 also appears to be a key coactivator for two distinct lineages of normal and malignant hematopoiesis, namely myelomonopoiesis and erythropoiesis.
3865 Mediator/TRAP complex, a master transcriptional coregulator, constitutes a subcomplex of RNA polymerase II holoenzyme, and integrates a wide variety of intracellular signals through specific interactions of activators with specific Mediator subunits. Among circa 30 subunits, MED1/TRAP220, in which two LXXLL nuclear receptor recognition motifs (aa.604-608; aa.645-649) are closely situated, was identified initially as a specific coactivator for nuclear receptors, and subsequently as such for GATA family activators as well. We previously demonstrated that MED1/TRAP220 plays an important role in RAR- and VDR-mediated myelomonocytic differentiation of HL-60 acute promyelocytic leukemia cells. MED1 was also proven to serve in the process of GATA1-mediated normal erythropoiesis as a GATA1-specific coactivator. In this study, we analyzed the mechanism of MED1 action in GATA1-mediated erythroid differentiation by use of K562 human erythroleukemic cells, because GATA1-mediated erythroid differentiation is efficiently induced with hemin in these cells. Expressions of Mediator subunits including MED1 were strongly induced during hemin-induced erythroid differentiation, together with induced expressions of GATA1-target and erythroid lineage-specific genes such as gamma-globin, beta-globin, porphobilinogen deaminase (PBGD) and 5-aminolevulinate synthase (ALAS-E), suggesting that MED1 (or Mediator) plays a role in this process. When MED1 in hemin-treated K562 cells was knocked down with siRNA, the number of cells positive for benzidine staining decreased, and expressions of GATA1-target and erythroid marker genes (above) were reduced, markedly. Inversely, when the full-length MED1 was overexpressed in these cells, the number of benzidine-positive cells increased, and the expressions of GATA1-target and erythroid marker genes enhanced, prominently. GST pulldown and mammalian two hybrid assays disclosed that MED1(aa.681-715) is responsible for GATA1 binding. Unexpectedly, overexpressions of MED1(aa.1-602) and MED1(aa.1-703), the N-terminal MED1 truncations that were devoid of the GATA1-binging ability, also enhanced the number of benzidine-positive cells and the expressions of GATA1-target and erythroid marker genes in a manner similar to the full-length MED1. Hence, a bypassed GATA1 signaling that escapes its interaction with MED1 appears to exist. Recently, a bypass molecule CCAR1 was reported to interact simultaneously with nuclear receptors and the N-terminus of MED1 and to form a bypass pathway that does not require the direct interaction between nuclear receptors and LXXLL motifs of MED1. We next asked if CCAR1 might also bypass the GATA1-MED1 pathway. To analyze the transcriptional mechanism, we performed luciferase reporter assays using the gamma-globin promoter in the wild-type mouse embryonic fibroblasts (MEFs). The reporter activity was enhanced in a GATA1-dose dependent manner. CCAR1 by itself could enhance the reporter activity. When a small dose of GATA1 was co-expressed, the reporter activity was further enhanced. However, even if p300 was overexpressed, the reporter activity was not enhanced any more. Thus, CCAR1, but not p300, appears to be involved in GATA1-mediated transcription in this system. Next, when various truncations of MED1 were added back into the Med1-/- MEFs, the N-terminal truncations, MED1(aa.1-602) and MED1(aa.1-703), enhanced the GATA1-mediated reporter activity, but the enhancement was not as great as the one when the full-length MED1 was added back. This difference might reflect the contribution of the GATA1 and MED1 direct interaction. GST-pulldown and mammalian two-hybrid assays showed that CCAR1 does interact with the C-terminal zinc-finger domain of GATA1. Further, ChIP assays showed that GATA1, MED1 and CCAR1 are all recruited onto the gamma-globin promoter. Taken together, these studies suggest that MED1 functions as a GATA1 specific coactivator not only in normal, but also in malignant, erythroid differentiation, and that a dual mechanism of GATA1-MED1 pathway exists in GATA1-mediated erythroid differentiation, namely, the direct interaction between MED1 and GATA1 and the bypassed interaction of these molecules via CCAR1. The dual mode of MED1-madiated coactivation in the GATA1 pathway might contribute to the fine tuning of the GATA1 function in erythropoiesis of a living animal. Disclosures: No relevant conflicts of interest to declare.
1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.
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