The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodeling factor Mi-2␣ as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2␣ and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2␣, previously studied as a subunit in the NuRD corepressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2␣ in c-Myb-expressing K562 cells, led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2␣ were compared, the Myb-Mi-2␣ co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2␣. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2␣, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4-tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2␣ transactivational co-operation, as did co-transfection with p300.
The c-Myb oncoprotein is a DNA-binding transcription factor with a key role in early stages of hematopoiesis. To expand our knowledge of partners cooperating with c-Myb, we performed a yeast two-hybrid screening with full-length c-Myb as bait. Here, we report FLICEassociated huge protein (FLASH)/CASP8AP2 as a novel Myb-interacting protein. We show that FLASH interacts with the DNA-binding domain of c-Myb and enhances c-Myb-dependent reporter activity and expression of endogenous c-Myb target genes. Chromatin immunoprecipitation assays revealed that FLASH and c-Myb both associate with the MYC promoter region as well as with the intronic enhancer of the c-Myb target gene ADA. Furthermore, siRNA knock-down of FLASH or c-Myb both result in a reduction of MYC and ADA expression. The co-activator effect is mediated through the C-terminal part of FLASH, which binds c-Myb. The FLASH-induced enhancement is comparable with the increase seen with the c-Myb co-activator p300. We find FLASH localized in discrete nuclear speckles in several cell lines, co-localized with c-Myb in active RNA polymerase II foci. These results imply a novel molecular mechanism of regulation of c-Myb activity. We propose that c-Myb cooperates with FLASH in foci associated with active RNA polymerase II, leading to enhancement of Myb-dependent gene activation.
BackgroundFLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator.ResultsTo find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories.ConclusionsWe conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci.
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