FLASH is an essential component of Cajal bodies

Barcaroli, Daniela; Dinsdale, David; Neale, Michael H.; Bongiorno-Borbone, Lucilla; Ranalli, Marco; Munarriz, Eliana; Sayan, Emre; McWilliam, J. M.; Smith, Tim; Fava, Eugenio; Knight, Richard; Melino, Gerry; Laurenzi, Vincenzo De

doi:10.1073/pnas.0604225103

Cited by 54 publications

(73 citation statements)

References 30 publications

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“…We therefore conclude that FLASH is predominantly nuclear, both when transfected into cells and when endogenously expressed. Very recently Barcaroli et al (2006b) and Milovic-Holm et al (2007) published evidence supporting the same conclusion.…”

Section: Discussionmentioning

confidence: 61%

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FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci

et al. 2008

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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.

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Section: Discussionmentioning

confidence: 61%

“…More likely, a partial co-localization simply reflects a dynamic association. Strong evidence for association between FLASH and Cajal bodies was recently reported by Barcaroli et al, (2006b). Association with PML nuclear bodies was also recently reported (Milovic-Holm et al, 2007).…”

Section: Discussionmentioning

confidence: 66%

FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci

et al. 2008

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“…On transcription or translation block, FLASH is rapidly degraded, whereas NPAT persists for longer (see ref. 15). Most importantly, depletion of NPAT results in a failure to enter S-phase (9), whereas down-regulation of FLASH blocks progression through S-phase (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Although FLASH was originally identified as a component of the apoptotic signaling complex known as the death-inducing signaling complex (DISC) that is assembled in response to Fas ligand binding (13,14), we have recently shown that FLASH is an essential component of CBs and is required for maintenance of their structure (15). We show that FLASH colocalizes with the histone transcriptional activator, NPAT, in CBs and is required for efficient expression of histone genes.…”

mentioning

confidence: 82%

FLASH is required for histone transcription and S-phase progression

Barcaroli

Bongiorno-Borbone

Terrinoni

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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116

135

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3). In particular, the U7 small nuclear ribonucleoproteins and other factors involved in histone precursor mRNA processing are known to accumulate within CBs (1, 4). Notably, CBs also associate with the major histone gene clusters in a variety of organisms, including mammals, amphibians, and dipterans (5, 6). In addition to participating in various RNA-processing activities, CBs have also been implicated in transcriptional regulation of the cell-cycledependent histone genes. Phosphorylation of a CB component p220͞nuclear protein, ataxia-telangiectasia (NPAT) by cyclin E͞Cdk2 is required for activation of histone transcription, exit from G 1 , and progression through S phase (7-12). Taken together, these observations suggest that CBs are intimately involved in histone gene expression.In this study, we identify FADD-like IL-1␤-converting enzyme (FLICE) associated huge protein (FLASH) (13) as a component of the histone gene expression machinery. Although FLASH was originally identified as a component of the apoptotic signaling complex known as the death-inducing signaling complex (DISC) that is assembled in response to Fas ligand binding (13, 14), we have recently shown that FLASH is an essential component of CBs and is required for maintenance of their structure (15). We show that FLASH colocalizes with the histone transcriptional activator, NPAT, in CBs and is required for efficient expression of histone genes. Results FLASH Down-Regulation Results in S-Phase Block.One of the hallmarks of proteins that are involved in expression of the cell-cycle-dependent histone genes is that perturbation of their function results in an accumulation of cells in S phase. Accordingly, we found that treatment of cells with short hairpin RNAs (shRNAs) targeting FLASH (shFLASH) resulted in a dramatic block of cells within S-phase of the cell cycle (Fig. 1a). Such a block was observed in all cell lines tested (HEK293, HeLa, MCF-7, SAOS2, 3T3 and MEFs) reaching up to 70% after 72 h (see Fig. 5, which is published as supporting information on the PNAS web site). These findings were confirmed through use of a colony-forming assay, revealing that down-regulation of FLASH resulted in a remarkable reduction in growth of the shFLASH-treated cells (Fig. 1b). Western blot in Fig. 1c confirms FLASH protein levels downregulation after shRNA treatment.Another hallmark of genes involved in histone gene expression is that their protein levels are up-regulated during S phase. Endogenous FLASH expression showed a clear cell-cycledependence, peaking during S-phase, when cells were synchronized by thymidine block and deoxycytidine release (Fig. 1d). Consistent with these observations, we found that the number of FLASH-positive bodies was correlated with the cell cycle. Primary (IMR90) cells were used for this analysis, as they are diploid. As shown in Fig. 1e, the number of FLASH bodies in BrdU-positive (S-phase cells) was typically four, whereas in BrdU-negative cells, the number was typically two. FLASH Interacts with NPAT and Is Bound to Histone Ge...

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“…More recently, FLASH was shown to localize to Cajal bodies and function in transcriptional control or RNA processing (68 -71). It is conceivable that the SBD in FLASH contributes to sumoylation-regulated assembly of nuclear complexes such as Cajal bodies or is responsible for the communication between Cajal body and sumoylated nuclear entities such as PML bodies (3,70,72).…”

Section: Volume 287 • Number 50 • December 7 2012mentioning

confidence: 99%

Poly-Small Ubiquitin-like Modifier (PolySUMO)-binding Proteins Identified through a String Search

Sun

Hunter

2012

Journal of Biological Chemistry

111

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Background: Sumoylation is recognized by proteins with SUMO-interacting motifs. Results: SUMO-interacting motifs were identified through a computational string search and validated in SUMO binding assays. Conclusion: Arkadia, FLASH, C5orf25, and SOBP all contain clustered SIMs. Significance: These proteins contain distinct SUMO binding structures responsible for the recognition of diverse forms of sumoylation.

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FLASH is an essential component of Cajal bodies

Cited by 54 publications

References 30 publications

FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci

FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci

FLASH is required for histone transcription and S-phase progression

Poly-Small Ubiquitin-like Modifier (PolySUMO)-binding Proteins Identified through a String Search

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