The long noncoding RNA NEAT1 and nuclear paraspeckles are up-regulated by the transcription factor HSF1 in the heat shock response

Abstract

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

Keywords: NEAT1; gene expression; heat shock factor protein 1 (HSF1); heat shock protein (HSP); heat shock response; long noncoding RNA (long ncRNA, lncRNA); paraspeckle; sulforaphane; transcription regulation.

Figure 1.

NEAT1 expression and paraspeckle formation are induced by SFN. A, MCF7 cells were treated with SFN (20 μm) for the indicated time points. RNA was isolated, and the expression of NEAT1 (both isoforms) and NEAT1_2 was determined by RT-qPCR. The mean value ± S.D. of three biological replicates in one experiment is presented as fold change relative to untreated cells. The results are representative of three independent experiments. B, MCF7 cells were pre-incubated with NAC (5 mm) and then treated with SFN for 6 h. NEAT1 expression was determined as described in A. C, MCF7 cells were left untreated or treated with SFN for 6 h, fixed, and subjected to RNA-FISH using probes recognizing the NEAT1_2 isoform. DAPI was used to visualize the nuclei. Bars, 10 μm. D, overall intensity of the dots in at least 250 cells was quantitated using the Volocity software. Mean values ± S.D. of three biological replicates are shown and presented as fold change relative to untreated cells. p values were calculated using ANOVA (A) or Student's t test (B and D) with p < 0.05 considered statistically significant. (***, p ≤ 0.001; **, p ≤ 0.01.)

Figure 2.

NEAT1 induction by SFN is not dependent on NRF2. A, MCF7 cells were transfected with an siRNA specifically targeting NRF2 (siNRF2) or control siRNA (siCtrl). Twenty four h post-transfection, cells were either left untreated or treated with SFN (20 μm) for 6 h. NEAT1 expression was determined by RT-qPCR as described in Fig. 1. Depletion of NRF2 expression in whole-cell extracts was verified by Western blotting analyses using an NRF2 antibody. The membrane was re-probed with an anti-actin antibody to ensure equal loading. B and C, MCF7 cells were transfected as described in A and subjected to a long-term treatment with SFN (10 μm) for 24 h. The expression of NEAT1 and NEAT1_2 (B) and NQO1 (C) was determined by RT-qPCR. Experiments were performed in triplicate, and the graph is representative of three independent experiments. (**, p ≤ 0.01; *, p, ≤ 0.05; ns, not significant.)

Figure 3.

SFN-induced NEAT1 expression depends on HSF1. A and B, MCF7 cells were left untreated or treated with SFN (20 μm) for 6 h. HSF1 expression in WCE (A) and nuclear extracts (NE) (B) were determined by immunoblot analyses. Equal loading was verified by re-probing the membranes with actin (A) or lamin B (B) antibodies. C, cells were treated with SFN as described above, and HSP70 expression was determined by RT-qPCR. D, MCF7 cells were transfected with two different siRNAs targeting HSF1, siHSF1_#1, and siHSF1_#2, or a control siRNA. Forty eight hours post-transfection, cells were left untreated or treated with SFN for 6 h. NEAT1 expression was assessed by RT-qPCR. SiRNA-mediated HSF1 depletion was verified by immunoblot analyses. E, HeLa cells were transfected with siHSF1_#2 or control siRNA, and after 48 h SFN-induced NEAT1 expression was determined by RT-qPCR. HSF1 expression was determined by immunoblot analyses using actin as loading control. (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.)

Figure 4.

HSF1 depletion abrogates SFN-induced paraspeckle formation. A, MCF7 cells were transfected with an HSF1-specific siRNA or a control siRNA. After 48 h, cells were left untreated or treated with SFN for 6 h, fixed, and subjected to coimmuno-FISH analyses by confocal microscopy using an antibody recognizing HSF1 (red) and fluorescent probes binding to NEAT1_2 (green). Nuclei were visualized with DAPI (blue). All experiments were performed in triplicate. Bars, 10 μm. B and C, intensities of NEAT1_2 containing paraspeckles and nuclear HSF1 staining in at least 250 cells were quantitated using Volocity software. (***, p ≤ 0.001.)

Figure 5.

HSF1 binds to and transcriptionally activates the NEAT1 promoter. A, MCF7 cells were transfected with a luciferase reporter vector containing 4040 bp of the NEAT1 upstream region (pNEAT1-luc) or empty control vector. After 24 h, cells were left untreated or treated with SFN (20 μm) for 8 h, and luciferase assays were performed. The experiments were performed in triplicate, and mean values ± S.D. are shown. The result is representative of three independent experiments. B, MCF7 cells were co-transfected with pNEAT1-luc and siHSF1_#2 as described under “Experimental procedures.” Cells were left untreated or treated with SFN for 8 h, and luciferase assays were performed. C, sequence conservation within NEAT1 upstream regions is illustrated by PhyloP Basewise Conservation scores from 100 vertebrates (USCS Genome Browser). An alignment of conserved HSE core sequences from human, rhesus, mouse, dog, and elephant is shown. D, truncated mutant of the NEAT1 promoter luciferase reporter construct encompassing the putative HSE site was generated and transfected into MCF7 cells along with a version harboring four point mutations within the HSE consensus sequence. SFN-induced luciferase activity was measured 24 h post-transfection. E, MCF7 cells were left untreated or treated with SFN (20 μm) for 6 h, and ChIP assays were performed using an anti-HSF1 antibody. qPCR was performed with primers flanking the HSE site. Primers flanking a region further upstream in the NEAT1 promoter (“upstr”), as well as primers amplifying a region of the GAPDH promoter, were used as negative controls. The relative amount of immunoprecipitated DNA compared with input DNA for each primer set is shown for the HSF1 ChIP. The values obtained by the IgG ChIP was less than 0.003% for the HSF1 and control primers. The result is representative of three independent ChIP experiments, where qPCR reactions were done as triplicates. (***, p ≤ 0.001; **, p ≤ 0.01; ns, not significant.)

Figure 6.

NEAT1 is induced by heat shock. A and B, MCF7 cells were subjected to heat shock by incubation at 43 °C for 30 min and then returned to 37 °C to recover for the indicated time periods. Activation of HSF1 was verified by shifted migration in Western blotting analyses (A) and by induction of HSP70 mRNA expression (B). C, cells were treated as above, and expression of NEAT1 and NEAT1_2 was assessed by RT-qPCR. (***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.)

Figure 7.

Proliferation is compromised in NEAT1-depleted cells. A, MCF7 cells were transfected with two LNA-GapmeR antisense oligos targeting NEAT1 or a negative control oligo and immediately placed in a IncuCyte® live cell analysis system for cell confluence monitoring. After 48 h, cells were removed from the incubator, and for half of the cells the medium was changed at 37 °C, whereas the other half was subjected to heat shock at 43 °C for 30 min. All the cells were then returned to the IncuCyte® live cell analysis system and monitored for another 48 h. Confluency (%) was calculated using the IncuCyte® S3 software. Mean values ± S.D. of 15 images (three images from each well of five wells in total) are shown. The results are representative for three independent experiments. B, relative confluency of cells over the last 48 h of the experiment described in A is shown.

Figure 8.

NEAT1 knockdown amplifies the expression of HSF1 target genes upon heat shock. MCF7 cells were transfected with two different LNA-GapmeR NEAT1 antisense oligos either targeting both isoforms of NEAT1 or solely the long NEAT1_2 isoform and a negative control oligo. After 48 h, cells were subjected to heat shock and recovered for the indicated time periods. The expression of Hsp70, Hsp90, and Hsp27 was determined by RT-qPCR. Knockdown of NEAT1 and NEAT1_2 was verified by RT-qPCR. (***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.)