Abstract
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.
Highlights
FLICE associated huge protein (FLASH) is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-B activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription
Consistent with the up-regulation of activity of both FLASH and c-Myb, all three proteins, FLASH, c-Myb and PIAS1, are co-localized in active RNA polymerase II foci. These results suggest that FLASH and PIAS1 cooperate in enhancing c-Myb transcriptional activity in foci that resemble transcription factories
This part of the protein contains the DED-recruiting domain (DRD) [3] and includes the region that interacts with c-Myb [6]
Summary
FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-B activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. 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. We showed that FLASH enhanced the expression of the endogenous c-Myb target gene mim-1 and knock-down of FLASH resulted in a reduction in expression of c-Myb target genes in haematopoietic cells [6]. All these diverse roles point to FLASH as being a multifunctional nuclear protein
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