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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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Sat III transcripts are very large transcripts inducibly transcribed from one strand during stress. (A) Reverse transcription was performed on total RNA extracts prepared from HeLa cells submitted or not to a 1-h heat shock at 42°C, with either an hsp70 antisense, a pHuR98 antisense (black), or a pHuR98 sense (gray) primer. The reaction products were spotted onto nitrocellulose membrane and quantitated. The sequence of the double-stranded pHuR98 clone is shown on the right. A weak signal is observed for hsp70, pHuR98 antisense, and pHuR98 sense transcripts at 37°C. Heat shock induces a ninefold increase in the level of hsp70 transcripts. Likewise, a ninefold increase in the intensity of the signal corresponding to pHuR98 sense transcripts is observed at 42°C, while the signal corresponding to pHuR98 antisense transcripts decreases by two. (B) A run-on assay was performed with nuclei prepared from cells submitted or not to a 1-h heat shock at 42°C. The following probes were used: pGEM2 (control for nonspecific hybridization), pH2.3 (hsp70 gene), pHuR98 (sat III), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene), which serves as a normalization control for transcription. Quantification of the spots by PhosphorImager® revealed that stress induces an 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. (C) A Northern blot analysis was performed on total RNA extracts prepared from HeLa cells. Different probes were used: an hsp70 probe (Wu et al., 1985), the pHuR98 clone, an antisense pHuR98 oligonucleotide to reveal sense transcripts (gray), or a sense pHuR98 oligonucleotide to reveal antisense transcripts (black). Ethidium bromide staining of the gel before transfer confirms that equal amounts of RNA extracts were loaded in each lane. For hsp70 transcripts, a very weak signal is observed for the 37°C sample, while a signal at the expected size (2.3 kb) is observed in the 42°C sample (the positions of the 28S and 18S rRNAs are indicated). For pHuR98 transcripts, a faint signal is present with all three probes at 37°C, while in the 42°C sample, a very slowly migrating band is revealed, both with the pHuR98 probe and the antisense oligonucleotide (gray). No signal is observed with the sense oligonucleotide (black).
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fig7: Sat III transcripts are very large transcripts inducibly transcribed from one strand during stress. (A) Reverse transcription was performed on total RNA extracts prepared from HeLa cells submitted or not to a 1-h heat shock at 42°C, with either an hsp70 antisense, a pHuR98 antisense (black), or a pHuR98 sense (gray) primer. The reaction products were spotted onto nitrocellulose membrane and quantitated. The sequence of the double-stranded pHuR98 clone is shown on the right. A weak signal is observed for hsp70, pHuR98 antisense, and pHuR98 sense transcripts at 37°C. Heat shock induces a ninefold increase in the level of hsp70 transcripts. Likewise, a ninefold increase in the intensity of the signal corresponding to pHuR98 sense transcripts is observed at 42°C, while the signal corresponding to pHuR98 antisense transcripts decreases by two. (B) A run-on assay was performed with nuclei prepared from cells submitted or not to a 1-h heat shock at 42°C. The following probes were used: pGEM2 (control for nonspecific hybridization), pH2.3 (hsp70 gene), pHuR98 (sat III), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene), which serves as a normalization control for transcription. Quantification of the spots by PhosphorImager® revealed that stress induces an 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. (C) A Northern blot analysis was performed on total RNA extracts prepared from HeLa cells. Different probes were used: an hsp70 probe (Wu et al., 1985), the pHuR98 clone, an antisense pHuR98 oligonucleotide to reveal sense transcripts (gray), or a sense pHuR98 oligonucleotide to reveal antisense transcripts (black). Ethidium bromide staining of the gel before transfer confirms that equal amounts of RNA extracts were loaded in each lane. For hsp70 transcripts, a very weak signal is observed for the 37°C sample, while a signal at the expected size (2.3 kb) is observed in the 42°C sample (the positions of the 28S and 18S rRNAs are indicated). For pHuR98 transcripts, a faint signal is present with all three probes at 37°C, while in the 42°C sample, a very slowly migrating band is revealed, both with the pHuR98 probe and the antisense oligonucleotide (gray). No signal is observed with the sense oligonucleotide (black).

Mentions: We were next interested in the characterization of sat III transcripts. To estimate the induction level induced by heat exposure, and to determine from which strand the transcripts are generated, we performed reverse transcription on total RNA extracts from non–heat-shocked and heat-shocked HeLa cells with the following primers: antisense pHuR98, sense pHuR98, or antisense hsp70 (corresponding to the major heat-induced gene). The reaction samples were then spotted onto nitrocellulose membrane and quantified using the PhosphorImager® system (normalized to the 37°C value for each primer). Results are presented in Fig. 7 A. We found that both sense (gray) and antisense (black) sat III transcripts as well as hsp70 transcripts were present at very low levels in the 37°C samples as compared with input. After a 1-h heat shock at 42°C, we observed a twofold decrease in the amount of antisense sat III transcripts and a ninefold increase in the amount of sense sat III transcripts as compared with the 37°C samples. Likewise, a ninefold increase in hsp70 transcripts was observed at 42°C. A run-on assay was also performed with nuclei prepared from stressed and unstressed cells (Fig. 7 B). This experiment showed that stress induces a 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. Sat III repeats are thus inducibly transcribed during stress, essentially from one strand.


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)

Sat III transcripts are very large transcripts inducibly transcribed from one strand during stress. (A) Reverse transcription was performed on total RNA extracts prepared from HeLa cells submitted or not to a 1-h heat shock at 42°C, with either an hsp70 antisense, a pHuR98 antisense (black), or a pHuR98 sense (gray) primer. The reaction products were spotted onto nitrocellulose membrane and quantitated. The sequence of the double-stranded pHuR98 clone is shown on the right. A weak signal is observed for hsp70, pHuR98 antisense, and pHuR98 sense transcripts at 37°C. Heat shock induces a ninefold increase in the level of hsp70 transcripts. Likewise, a ninefold increase in the intensity of the signal corresponding to pHuR98 sense transcripts is observed at 42°C, while the signal corresponding to pHuR98 antisense transcripts decreases by two. (B) A run-on assay was performed with nuclei prepared from cells submitted or not to a 1-h heat shock at 42°C. The following probes were used: pGEM2 (control for nonspecific hybridization), pH2.3 (hsp70 gene), pHuR98 (sat III), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene), which serves as a normalization control for transcription. Quantification of the spots by PhosphorImager® revealed that stress induces an 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. (C) A Northern blot analysis was performed on total RNA extracts prepared from HeLa cells. Different probes were used: an hsp70 probe (Wu et al., 1985), the pHuR98 clone, an antisense pHuR98 oligonucleotide to reveal sense transcripts (gray), or a sense pHuR98 oligonucleotide to reveal antisense transcripts (black). Ethidium bromide staining of the gel before transfer confirms that equal amounts of RNA extracts were loaded in each lane. For hsp70 transcripts, a very weak signal is observed for the 37°C sample, while a signal at the expected size (2.3 kb) is observed in the 42°C sample (the positions of the 28S and 18S rRNAs are indicated). For pHuR98 transcripts, a faint signal is present with all three probes at 37°C, while in the 42°C sample, a very slowly migrating band is revealed, both with the pHuR98 probe and the antisense oligonucleotide (gray). No signal is observed with the sense oligonucleotide (black).
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fig7: Sat III transcripts are very large transcripts inducibly transcribed from one strand during stress. (A) Reverse transcription was performed on total RNA extracts prepared from HeLa cells submitted or not to a 1-h heat shock at 42°C, with either an hsp70 antisense, a pHuR98 antisense (black), or a pHuR98 sense (gray) primer. The reaction products were spotted onto nitrocellulose membrane and quantitated. The sequence of the double-stranded pHuR98 clone is shown on the right. A weak signal is observed for hsp70, pHuR98 antisense, and pHuR98 sense transcripts at 37°C. Heat shock induces a ninefold increase in the level of hsp70 transcripts. Likewise, a ninefold increase in the intensity of the signal corresponding to pHuR98 sense transcripts is observed at 42°C, while the signal corresponding to pHuR98 antisense transcripts decreases by two. (B) A run-on assay was performed with nuclei prepared from cells submitted or not to a 1-h heat shock at 42°C. The following probes were used: pGEM2 (control for nonspecific hybridization), pH2.3 (hsp70 gene), pHuR98 (sat III), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene), which serves as a normalization control for transcription. Quantification of the spots by PhosphorImager® revealed that stress induces an 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. (C) A Northern blot analysis was performed on total RNA extracts prepared from HeLa cells. Different probes were used: an hsp70 probe (Wu et al., 1985), the pHuR98 clone, an antisense pHuR98 oligonucleotide to reveal sense transcripts (gray), or a sense pHuR98 oligonucleotide to reveal antisense transcripts (black). Ethidium bromide staining of the gel before transfer confirms that equal amounts of RNA extracts were loaded in each lane. For hsp70 transcripts, a very weak signal is observed for the 37°C sample, while a signal at the expected size (2.3 kb) is observed in the 42°C sample (the positions of the 28S and 18S rRNAs are indicated). For pHuR98 transcripts, a faint signal is present with all three probes at 37°C, while in the 42°C sample, a very slowly migrating band is revealed, both with the pHuR98 probe and the antisense oligonucleotide (gray). No signal is observed with the sense oligonucleotide (black).
Mentions: We were next interested in the characterization of sat III transcripts. To estimate the induction level induced by heat exposure, and to determine from which strand the transcripts are generated, we performed reverse transcription on total RNA extracts from non–heat-shocked and heat-shocked HeLa cells with the following primers: antisense pHuR98, sense pHuR98, or antisense hsp70 (corresponding to the major heat-induced gene). The reaction samples were then spotted onto nitrocellulose membrane and quantified using the PhosphorImager® system (normalized to the 37°C value for each primer). Results are presented in Fig. 7 A. We found that both sense (gray) and antisense (black) sat III transcripts as well as hsp70 transcripts were present at very low levels in the 37°C samples as compared with input. After a 1-h heat shock at 42°C, we observed a twofold decrease in the amount of antisense sat III transcripts and a ninefold increase in the amount of sense sat III transcripts as compared with the 37°C samples. Likewise, a ninefold increase in hsp70 transcripts was observed at 42°C. A run-on assay was also performed with nuclei prepared from stressed and unstressed cells (Fig. 7 B). This experiment showed that stress induces a 11.9-fold and a 13.4-fold induction in sat III and hsp70 transcription, respectively. Sat III repeats are thus inducibly transcribed during stress, essentially from one strand.

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