FLASH is required for histone transcription and S-phase progression

Abstract

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1beta-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.

Fig. 1.

Down-regulation of FLASH results in S-phase block. (a) Cell-cycle distribution of HeLa cells transfected with GFP-spectrin and either pSUPER-scrambled (control) or pSUPER-FLASH-1 (shFLASH). Cells were stained with PI, and GFP-positive cells were analyzed by flow cytometry for DNA content. Percentages of cells in G₁, S, or G₂/M calculated by using ModFit program are indicated. Identical results were obtained by using pSUPER-FLASH-2 (data not shown). (b) Colony-forming assay of SAOS-2 cells transfected with pBabe-puro and either pSUPER-scrambled (control) or pSUPER-FLASH-1 (shFLASH), selected for 2 weeks with 1 μg/ml puromycin. Cells transfected with pSUPER-FLASH-1 show a large reduction in colony numbers. (c) Western blot showing that transfection of MCF-7 cells with pSUPER-FLASH-1 (shFL), but not with a scrambled vector (control), results in reduction of FLASH protein levels. (d Upper) Western blot showing FLASH expression in MCF-7 cells untreated (control) or treated with either 2 mM thymidine for 16 h (G₁), followed by 4-h release in 24 μM deoxycytidine (S), or treated with 50 ng/ml nocodazole for 16 h (G₂). As a positive control, cells transfected with GFP-FLASH (FLASH) were loaded. The Western blot was reprobed with tubulin to show equal loading. (d Lower) Cell-cycle distribution of an aliquot of cells used for the Western blot. (e) Percentage of IMR90 cells containing 0, 1, 2, or 3–4 FLASH bodies positive (blue) or negative (red) for BrdU incorporation. The number of FLASH bodies per cell increases in S phase cells as reported for CBs.

Fig. 2.

FLASH interacts with p220/NPAT and binds to histone gene promoters. (a) Coimmunostaining by using anti-FLASH (green) and anti-NPAT (red) antibodies in SAOS-2 cells shows that the endogenous proteins colocalize (Scale bar = 5 μm). (b) Chromatin IP showing that FLASH binds to promoter sequences for histones: H2B/r, H2B/h, H3/c, and H4/e. Cells were transfected with either an empty vector (pCDNA3), with HA-FLASH (HA-FLASH), or with a vector coding for an unrelated HA-tagged protein (HA-p21). PCR was performed on DNA extracted from lysates subjected to IP with anti-HA antibody (lane 1) or with an unrelated anti-tubulin antibody (lane 2). As a positive control, PCR was performed on DNA extracted from untreated cells (lane 3). A positive PCR band appears only in HA-FLASH-transfected cells immunoprecipitated with anti-HA antibody but not in control-transfected or control-immunoprecipitated samples. (c) Luciferase assay on H1299 cells transfected with a luciferase reporter gene under the control of histone H4/e promoter sequence together with an empty vector (pCDNA) or HA-FLASH (FLASH). (d) HEK-293 cells were transfected with pEGFP or GFP-FLASH, and nuclear extracts were subjected to IP with anti-GFP antibody. Western blot against NPAT shows that endogenous NPAT coimmunoprecipitates with GFP-FLASH.

Fig. 3.

FLASH down-regulation affects histone gene mRNA and protein levels. (a) Representative RT-PCR showing mRNA steady state levels for histones H2A/p, H2B/j, H3/c, and H4/k in HeLa cells transfected with pGFP-spectrin and either pSUPER-scrambled (control) or pSUPER-FLASH-1 (shFLASH) (1:5 ratio). GFP-positive cells were sorted by flow cytometry 48 h after transfection, and total RNA was extracted. Twenty-four cycles of amplification were performed after checking that this reaction was in the linear amplification range. GAPDH amplification (G) was used to demonstrate equal loading. Down-regulation of FLASH results in a significant reduction of mRNA levels for all of the histones tested. HeLa cells were transfected with pGFP-spectrin and either pSUPER-scrambled (C) or pSUPER-FLASH-1 (I) (1:5 ratio), and harvested for protein and RNA extraction 24, 36, 48, and 72 h after transfection. Representative RT-PCR (b) and Western blot (c) showing mRNA and protein steady state levels for histone H4 (Upper). For the PCR, 24 cycles of amplification were performed after checking that this reaction is still in the linear range. GAPDH amplification was used to demonstrate equal loading, and tubulin was used to confirm equal loading in the Western blot (Lower). An aliquot of cells from the same experiment was used to analyze cell-cycle distribution of the cells, and the results are reported below the Western blot. (d) Immunofluorescence of MCF-7 cells transfected with pSUPER FLASH (shFLASH) together with pGFP-spectrin (5:1 ratio) and stained with anti-NPAT antibodies. (e) Immunofluorescence of coilin −/− MEFs by using antibodies against FLASH (red) p220/NPAT (green) shows that the protein still colocalize. (f) Western blot of MCF-7 cells transfected with pSUPER-coilin (shCoil) or with a control vector (C) and collected 48 h after transfection, using an anti-coilin antibody. A strong reduction of coilin protein levels is observed. (g) Immunofluorescence using antibodies against p220/NPAT (green) and FLASH (red) showing that in MCF-7 cells in which coilin has been down-regulated by transfection with pSUPER-coilin (shCoil) together with pGFP-spectrin for 48 h (first panel; green), and FLASH and NPAT still colocalize. Please note that, to use four colors, NPAT was stained with a secondary antibody emitting in far red and falsely colored in green to show the overlap with FLASH in red.

Fig. 4.

Down-regulation of coilin does not affect cell-cycle and histone transcription. HeLa cells were transfected with either a pSUPER-FLASH-1 (shFL) or pSUPER-coilin (shCoil) or pSUPER-scrambled sequence (Cont) together with pGFP-spectrin. An aliquot of each sample was used for cell-cycle detection, mRNA, and protein extraction. (a) Western blot for the indicated proteins was performed on HeLa cells treated as described earlier for 72 h. Whereas FLASH down-regulation results in the decrease of histones H4 and H3 and NPAT levels, coilin down-regulation produces only a very modest effect on histone H4 protein levels. (b) RT-PCR for histones H4/k and H3/c using mRNA of HeLa cells treated as described earlier for 48 and 72 h. After 72 h, down-regulation of coilin produces a modest effect on histone genes mRNA levels. (c) Cell-cycle analysis of cells transfected as described earlier for 72 h. Coilin down-regulation does not affect cell-cycle regulation.