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...

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3'-untranslated region (3'UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3'UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

Cajal Bodies are one of many specialised organelles contained in the eukaryotic cell nucleus, and are involved in a number of functions, including regulation of replication-dependent histone gene transcription. In normal diploid cells their number varies between 0 and 4 depending on the cell cycle phase, although in cancer cell lines their number is extremely variable and it has been suggested that it correlates with cell ploidy. Here we show that in mammalian cells, as in Drosophila, two distinct though functionally related bodies exist: a histone gene locus body and a Cajal Body. The first one can be detected using FLASH or NPAT as markers while the second is labelled using antibodies against Coilin. Only the number of FLASH/NPAT histone gene locus bodies correlates with ploidy and only these organelles appear to be regulated during the cell cycle. Finally, we show that the two organelles completely co-localize during the S phase of the cell cycle.

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.coiled bodies ͉ nuclear organelles C ajal bodies (CBs) are small nuclear organelles described in vertebrate cells a century ago by Ramon y Cajal and which have since been observed in a variety of animal and plant nuclei. Many components of CBs are shared with the nucleolus, and CBs frequently localize to the nucleolar periphery or within the nucleoli (1-3). CBs disappear from prophase nuclei and reappear in late G 1 after resumption of transcription in the daughter nuclei (for review see refs. 1, 4, and 5). Although their function is still in part elusive, recent work suggests that they are involved in several nuclear functions, including modification of small nuclear RNAs and small nuclear ribonucleoproteins, important for spliceosome formation, and assembly of the three eukaryotic RNA polymerases (pol I, pol II, and pol III) with their respective transcription and processing factors that are then transported as multiprotein complexes to the sites of transcription (1). More recently CBs have been implicated in replication-dependent histone gene transcription and mRNA maturation (1, 6-10), and a subset of CBs is physically associated with histone gene clusters on chromosomes 1 and 6 (11). Here we identify FLASH (FLICEassociated huge protein) (12) as a new component of CBs and show that it is essential for their structure.FLASH was initially identified as a component of the apoptosis signaling complex known as the death-inducing signaling complex (12, 13), which is associated with caspase 8 in the death-inducing signaling complex and thus essential for caspase 8 activation. However, this role of FLASH has been questioned (14). More recently it has been shown that, in response to TNF␣, FLASH translocates to the nucleus and binds the glucocorticoid receptor-interacting protein (GRIP-1), inhibiting both its interaction with, and the transcriptional activity of, the glucocorticoid receptor (15, 16). Results FLASH Has a Nuclear Localization.Despite the presence of three nuclear localization signals FLASH was originally described as a cytoplasmic protein (12,17). Staining endogenous FLASH with four different anti-FLASH antibodies, however, showed that FLASH only localized to the nucleus with a clear punctate appearance (Fig. 5a, which is published as supporting information on the PNAS web site). The specificity of the antibodies used was confirmed by the disappearance of the staining after short hairpin RNA for FLASH (Fig. 4a). West...