Hsp70 gene association with nuclear speckles is Hsp70 promoter specific - PubMed (original) (raw)

Hsp70 gene association with nuclear speckles is Hsp70 promoter specific

Yan Hu et al. J Cell Biol. 2010.

Abstract

Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid-processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association.

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Figures

Figure 1.

Transgene nuclear speckle association is specific for Hsp70 BAC. (A) BAC constructs (top to bottom): DHFR 057L22-K-8.32-C-29, Hsp70 92G8-K/N-8.32-C-2, and MT 134B2-K/N-8.32-C-8. Inducible genes whose expression we monitored (red), other genes (gray), transposable elements carrying lac operator repeats (green) and selectable markers (yellow), and vector backbones (blue) are shown in linear form. Arrows show transcription units. (B) Transgenes labeled with EGFP-LacI (green) showed no obvious association with nuclear speckles immunostained with SC-35 antibody (red) in four DHFR BAC and three MT BAC cell lines before (control [ctrl], top) and after (zinc, second row) zinc induction. All five Hsp70 cell lines showed increased nuclear speckle association 30 min after heat shock (H.S., bottom) relative to before heat shock (ctrl, third row). Optical sections are shown without deconvolution. Bar, 2 µm. (C) Nuclear speckle association for all BAC cell lines. DHFR and MT BAC cell lines showed a low background level of ∼10–20% speckle association. All Hsp70 BAC cell lines showed ∼40% elevated speckle associations before heat shock that increased significantly (P < 0.01) after heat shock. Error bars represent SEM.

Figure 2.

2.8-kb HSPA1A fragment is sufficient for nuclear speckle association. (A) Plasmid pSP14-14 contains the 2.8-kb HSPA1A gene and a 64-mer lac operator repeat. (B) RNA FISH and SC-35 immunostaining for pSP14_4_B4 CHO DG44 cell clone containing HSPA1A plasmid transgenes. (left) Nuclear DNA counterstained with DAPI (gray) and EGFP-LacI (green). Top two panels show HSPA1A RNA FISH signals (red) before (left) and after (right) heat shock. Images are shown without deconvolution. Bottom two panels show SC-35 staining (red) before (left) and after (right) heat shock. (right) Examples of RNA FISH combined with SC-35 immunostaining: EGFP-LacI (blue), SC-35 staining (green), and RNA FISH signals (red). Transgene arrays are shown at a higher magnification on the right. Bars, 2 µm. (C) Nuclear speckle association indexes for six independently derived cell clones. ctrl, control. h.s., 30 min after heat shock. Error bars show SEM.

Figure 3.

The HSPA1A transgene association with nuclear speckles is promoter dependent. (A, top left) Plasmid pSP14-MT2A + HSPA1A containing the MT2A promoter driving the HSPA1A gene and a 64-mer lac operator repeat. (bottom left) Transgene nuclear speckle association indexes for five independent cell clones before (ctrl) or after zinc induction. (right, top row) RNA FISH (red) before (left) and after (right) zinc induction; DAPI DNA counterstaining (blue). (right, bottom row) SC-35 staining (red) before (left) and after (right) zinc induction. (B) The corresponding layout as in A, but for plasmid pSP14-HSPpro + MT2A containing the HSPA1A promoter driving the MT2A gene. (C) Combined RNA FISH SC-35 immunostaining for cell clone pSP14-HSPpro + MT2A_C1. Transgenes were labeled with EGFP-LacI (left), RNA FISH signal (second from left), SC-35 staining (second from right), and merged image (right). Bars, 2 µm. Error bars show SEM.

Figure 4.

Transcription is required for nuclear speckle association. 30-min heat shock experiments using pSP14_4_B4 cells. Insets show enlarged regions containing the transgene array. (A) DAPI DNA staining is shown in blue, and EGFP-LacI is shown in green. (top) SC-35 staining (red) after heat shock (HS; left) or after heat shock with prior transcriptional inhibition with α-amanitin (right). (bottom) RNA FISH using HSPA1A probe (red) after heat shock (left) or α-amanitin treatment for 3.5 h before 30-min heat shock (right). (B) Nuclear speckle association indexes with or without transcriptional inhibitors. Bar, 2 µm. Error bars show SEM.

Figure 5.

The HSPA1A transgene nuclear speckle association does not correlate with levels of accumulated nascent transcripts. (A) Low copy integration of Hsp70 BAC recombineered to contain just one Hsp70 gene still shows speckle association despite small RNA FISH signal. RNA FISH (left, red) and SC-35 immunostaining (middle and right, red) for cell clone 69 containing 92G8_HSPA1B BAC. EGFP-LacI staining is shown in green, and DAPI DNA counterstaining is shown in gray. (left) After heat shock. (middle) No heat shock. (right) 30-min heat shock. (B) Nuclear speckle association indexes for three independent stable cell clones containing low copy integrations of the 92G8_HSPA1B BAC before (CTRL) and after (HS) heat shock. (C) RNA FISH of different cell lines, scaled uniformly, after gene activation. All hybridizations used the same HSPA1A probe and were performed in parallel. Top panel shows merged images of RNA FISH (red), EGFP-LacI (green), and DAPI (blue). Bottom panel shows RNA FISH signal only. Comparisons are between cell lines HSPA1B_69 (left) containing low copy integration of the Hsp70 BAC with one Hsp70 gene, pSP14_4_B4, with an HSPA1A plasmid array after heat shock (middle left) or cadmium (middle right) induction, and MTpro_HSPA1A_32_24 containing a plasmid array with the MT promoter driving a HSPA1A transcript after zinc addition (right). Numbers in bottom right corners are the integrated RNA FISH signal (top) or the RNA FISH mean intensity (bottom) per square micrometer. (D and E) Statistical analysis of the integrated RNA FISH signal intensities (D) or mean intensities per square micrometer (blue bars, E) versus nuclear speckle association indexes (red lines) for different cell lines under different transcription induction conditions as indicated in the figure. The cell line and induction conditions are indicated on the x axis: HspA1B_38_HS, HspA1B_69_HS, two clones of Hsp70 BAC with one Hsp70 gene after heat shock, pSP14_4_B4 after heat shock (4_B4_HS), pSP14_4_B4 after heat shock plus cadmium (4_B4_CD + HS), MTpro_HSPA1A_32_24 clone after zinc induction (MTpro_HSPA1A_32_24_Zn), and pSP14_4B4 after cadmium (4B4_Cd). Numbers shown below the table in blue are means for the integrated RNA FISH signal intensities (D) or mean intensities (E), and those shown in red are speckle association indexes. Bars, 2 µm. Cell numbers (n) are shown for each data point. Error bars represent SEM.

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Hsp70 gene association with nuclear speckles is Hsp70 promoter specific - PubMed (original) (raw)