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Stress-induced transcription of satellite III repeats.

Jolly C, Metz A, Govin J, Vigneron M, Turner BM, Khochbin S, Vourc'h C - J. Cell Biol. (2003)

Bottom Line: These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes.In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis.This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

View Article: PubMed Central - PubMed

Affiliation: INSERM U309, Institut A. Bonniot, 38706 La Tronche cedex, France. caroline.jolly@ujf-grenoble.fr

ABSTRACT
Exposure of mammalian cells to stress induces the activation of heat shock transcription factor 1 (HSF1) and the subsequent transcription of heat shock genes. Activation of the heat shock response also correlates with a rapid relocalization of HSF1 within a few nuclear structures termed nuclear stress granules. These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes. In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

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The heterochromatin protein HP1β is not concentrated in the 9q12 locus. HP1β-GFP construct (green) was transiently expressed in HeLa cells and codetected with either the DBD-TRIM mutant at 37°C or the endogenous HSF1 at 42°C (red). Projections of confocal series are shown. In both cases, nuclear stress granules are juxtaposed to, but never coincident with, HP1β-enriched foci (10 nuclei analyzed for each case). Insert shows a magnification of one stress granule. Bar, 5 μm.
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fig5: The heterochromatin protein HP1β is not concentrated in the 9q12 locus. HP1β-GFP construct (green) was transiently expressed in HeLa cells and codetected with either the DBD-TRIM mutant at 37°C or the endogenous HSF1 at 42°C (red). Projections of confocal series are shown. In both cases, nuclear stress granules are juxtaposed to, but never coincident with, HP1β-enriched foci (10 nuclei analyzed for each case). Insert shows a magnification of one stress granule. Bar, 5 μm.

Mentions: Based on these observations, we sought to determine if transcription occurs within the granules. Nuclear stress granules form primarily on the 9q12 juxtacentromeric region in humans, with putative secondary sites on chromosomes 12 and 15 and perhaps other chromosomes (Denegri et al., 2002; unpublished data). As the only characterized target sequence for HSF1 within the granules is a sat III repeat of the 9q12 region characterized by the clone pHuR98 (Jolly et al., 2002), we investigated the possibility that these repeats could be inducibly transcribed during stress. We first performed FISH to detect transcripts with the clone pHuR98 as a probe (Grady et al., 1992). Results are presented in Fig. 4. Both at 37°C and 42°C, a diffuse nucleoplasmic and cytoplasmic staining was observed (Fig. 4 A). In addition, at 42°C, three to four bright nuclear foci were also visible in most cells. These granular foci corresponded to transcripts as confirmed by their absence in cells treated with RNase A before hybridization (Fig. 4 A). The intensity of the nuclear and cytoplasmic diffuse staining is comparable to the background level both at 37°C and 42°C, thus demonstrating that sat III transcripts are essentially concentrated in the three to four nuclear foci (Fig. 4 B). Similarly RNA FISH experiments performed with probes corresponding to either chromosome 9 classical satellites (D9Z1), chromosome 9 centromeric repeats, or chromosome 16 sat II repeats (pHuR195) did not reveal any signal (not depicted). Codetection of sat III transcripts with HSF1 or RNA pol II showed a colocalization of these transcript foci with nuclear stress granules (Fig. 4 C). Finally, to confirm that the presence of three to four transcript foci was reflecting the number of copies of the 9q12 locus in HeLa cells, we also performed codetection of sat III transcripts with chromosome 9 centromeric repeats revealed by DNA FISH. As shown in Fig. 4 C, each transcript focus was found in the vicinity of a centromere, confirming that each RNA FISH signal corresponded to sat III transcripts emerging from one chromosome 9. Altogether, these observations show that heat shock specifically induces the transcription of sat III repeats from the three to four 9q12 loci present in HeLa cells. Interestingly, the 9q12 region has been described as heterochromatic based on its enrichment in methylcytosine-rich DNA (Miller et al., 1974). To better determine the chromatin flavour of the 9q12, we investigated the distribution of one of the best characterized markers of heterochromatin: the heterochromatin protein HP1β (Eissenberg and Elgin, 2000). Cells were transiently transfected with an HP1-GFP construct, and nuclear stress granules were revealed either by a mutant HSF1 (DBD+TRIM), which forms constitutive granules at 37°C (Jolly et al., 2002), or by the endogenous HSF1 at 42°C. As shown in Fig. 5, stress granules were always juxtaposed to an HP1β-rich focus, which corresponded to the close centromere of chromosome 9, but never enriched in HP1β protein, demonstrating that the juxtacentromeric 9q12 region does not display conventional heterochromatic features.


Stress-induced transcription of satellite III repeats.

Jolly C, Metz A, Govin J, Vigneron M, Turner BM, Khochbin S, Vourc'h C - J. Cell Biol. (2003)

The heterochromatin protein HP1β is not concentrated in the 9q12 locus. HP1β-GFP construct (green) was transiently expressed in HeLa cells and codetected with either the DBD-TRIM mutant at 37°C or the endogenous HSF1 at 42°C (red). Projections of confocal series are shown. In both cases, nuclear stress granules are juxtaposed to, but never coincident with, HP1β-enriched foci (10 nuclei analyzed for each case). Insert shows a magnification of one stress granule. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171959&req=5

fig5: The heterochromatin protein HP1β is not concentrated in the 9q12 locus. HP1β-GFP construct (green) was transiently expressed in HeLa cells and codetected with either the DBD-TRIM mutant at 37°C or the endogenous HSF1 at 42°C (red). Projections of confocal series are shown. In both cases, nuclear stress granules are juxtaposed to, but never coincident with, HP1β-enriched foci (10 nuclei analyzed for each case). Insert shows a magnification of one stress granule. Bar, 5 μm.
Mentions: Based on these observations, we sought to determine if transcription occurs within the granules. Nuclear stress granules form primarily on the 9q12 juxtacentromeric region in humans, with putative secondary sites on chromosomes 12 and 15 and perhaps other chromosomes (Denegri et al., 2002; unpublished data). As the only characterized target sequence for HSF1 within the granules is a sat III repeat of the 9q12 region characterized by the clone pHuR98 (Jolly et al., 2002), we investigated the possibility that these repeats could be inducibly transcribed during stress. We first performed FISH to detect transcripts with the clone pHuR98 as a probe (Grady et al., 1992). Results are presented in Fig. 4. Both at 37°C and 42°C, a diffuse nucleoplasmic and cytoplasmic staining was observed (Fig. 4 A). In addition, at 42°C, three to four bright nuclear foci were also visible in most cells. These granular foci corresponded to transcripts as confirmed by their absence in cells treated with RNase A before hybridization (Fig. 4 A). The intensity of the nuclear and cytoplasmic diffuse staining is comparable to the background level both at 37°C and 42°C, thus demonstrating that sat III transcripts are essentially concentrated in the three to four nuclear foci (Fig. 4 B). Similarly RNA FISH experiments performed with probes corresponding to either chromosome 9 classical satellites (D9Z1), chromosome 9 centromeric repeats, or chromosome 16 sat II repeats (pHuR195) did not reveal any signal (not depicted). Codetection of sat III transcripts with HSF1 or RNA pol II showed a colocalization of these transcript foci with nuclear stress granules (Fig. 4 C). Finally, to confirm that the presence of three to four transcript foci was reflecting the number of copies of the 9q12 locus in HeLa cells, we also performed codetection of sat III transcripts with chromosome 9 centromeric repeats revealed by DNA FISH. As shown in Fig. 4 C, each transcript focus was found in the vicinity of a centromere, confirming that each RNA FISH signal corresponded to sat III transcripts emerging from one chromosome 9. Altogether, these observations show that heat shock specifically induces the transcription of sat III repeats from the three to four 9q12 loci present in HeLa cells. Interestingly, the 9q12 region has been described as heterochromatic based on its enrichment in methylcytosine-rich DNA (Miller et al., 1974). To better determine the chromatin flavour of the 9q12, we investigated the distribution of one of the best characterized markers of heterochromatin: the heterochromatin protein HP1β (Eissenberg and Elgin, 2000). Cells were transiently transfected with an HP1-GFP construct, and nuclear stress granules were revealed either by a mutant HSF1 (DBD+TRIM), which forms constitutive granules at 37°C (Jolly et al., 2002), or by the endogenous HSF1 at 42°C. As shown in Fig. 5, stress granules were always juxtaposed to an HP1β-rich focus, which corresponded to the close centromere of chromosome 9, but never enriched in HP1β protein, demonstrating that the juxtacentromeric 9q12 region does not display conventional heterochromatic features.

Bottom Line: These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes.In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis.This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

View Article: PubMed Central - PubMed

Affiliation: INSERM U309, Institut A. Bonniot, 38706 La Tronche cedex, France. caroline.jolly@ujf-grenoble.fr

ABSTRACT
Exposure of mammalian cells to stress induces the activation of heat shock transcription factor 1 (HSF1) and the subsequent transcription of heat shock genes. Activation of the heat shock response also correlates with a rapid relocalization of HSF1 within a few nuclear structures termed nuclear stress granules. These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes. In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

Show MeSH
Related in: MedlinePlus