FLASH is required for histone transcription and S-phase progression

Abstract

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...

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

“…As shown in Figure 8d, we found that c-Myb and FLASH bound to the promoter region of the MYC gene harbouring a strong c-Myb-responsive element (Schmidt et al, 2000) as well as to the c-Myb responsive intronic enhancer of the ADA gene (Ess et al, 1995). As a positive reference for FLASH ChIP we used binding to the histone H3 promoter previously reported to be occupied by and regulated by FLASH (Barcaroli et al, 2006a). The enrichment of FLASH measured on MYC and ADA were very close to what we observed on the histone H3 promoter (Figure 8d).…”

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

“…The current data strongly suggest that at least a subset of Cajal bodies contains an integrated supramolecular architectural complex in which histone gene transcription factors, the coactivator p220 NPAT , histone gene clusters and the U7 snRNP related 3 0 end processing machinery are all associated contemporaneously. Recent results suggest that p220 NPAT and FLASH are necessary to maintain this structure during the cell cycle (Ye et al, 2003;Barcaroli et al, 2006).…”

ABCE1 is essential for S phase progression in human cells

“…71,72 Indeed, as demonstrated by several studies, the transcriptional repression of histone mRNA or depletion of the essential histone mRNA regulators in mammalian cells result in inhibition of DNA synthesis and S phase progression arrest. 53,[73][74][75] A similar effect has been shown to be caused by the inactivation of chromatin assembly system, which was also accompanied by DNA damage accumulation. 76,77 In order to check whether ABCE1 silencing affects histone biosynthesis, we analyzed the steady-state levels of two core histone proteins, H2B and H4, as well as the expression of their mRNAs during S phase, and those appeared to be significantly reduced ( Fig.…”