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Fragile X (CGG)n repeats induce a transcriptional repression in cis upon a linked promoter: evidence for a chromatin mediated effect.

Chandler SP, Kansagra P, Hirst MC - BMC Mol. Biol. (2003)

Bottom Line: In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte.We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length.Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sangamo BioSciences, 501 Canal Blvd, Ste A100, Point Richmond Tech Center II, Richmond, CA 94804, USA. schandler@sangamo.com

ABSTRACT

Background: Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture.

Results: In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin maturation and, using in vitro transcription, we show that chromatin formation is a fundamental part of the repressive pathway mediated by (CGG)n repeats. Using Trichostatin A, a histone deacetylase inhibitor, we show reactivation of the silenced promoter.

Conclusions: Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.

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The Role of Chromatin in Repeat Induced Transcriptional Repression (a) Primer extension products are shown from run-off RNA transcripts performed in vitro using a Hela cell nuclear extract incubated with pHSVtkCAT, in vitro CpG methylated pHSVtkCAT, pHSVtkCAT-(CGG)27, pHSVtkCAT-(CGG)70 and pHSVtkCAT-(CGG)105 DNAs. All reactions included control of pCMV-CAT DNA. (b) A graphical representation of the detectable transcript in the Helascribe reaction. The transcription was normalised to CMV-CAT as an internal control. (c) Primer extension analysis of the pHSVtkCAT-(CGG)140 reporter and pCMV-CAT injection control. Injections were of 5 ng reporter and 0.3 ng injection control per oocyte. The injected oocyte populations were incubated for 18 hours in the presence of 30 nM TSA. The primer extension products are shown with arrows and include the endogenous H4 message as an mRNA preparation control.
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Figure 4: The Role of Chromatin in Repeat Induced Transcriptional Repression (a) Primer extension products are shown from run-off RNA transcripts performed in vitro using a Hela cell nuclear extract incubated with pHSVtkCAT, in vitro CpG methylated pHSVtkCAT, pHSVtkCAT-(CGG)27, pHSVtkCAT-(CGG)70 and pHSVtkCAT-(CGG)105 DNAs. All reactions included control of pCMV-CAT DNA. (b) A graphical representation of the detectable transcript in the Helascribe reaction. The transcription was normalised to CMV-CAT as an internal control. (c) Primer extension analysis of the pHSVtkCAT-(CGG)140 reporter and pCMV-CAT injection control. Injections were of 5 ng reporter and 0.3 ng injection control per oocyte. The injected oocyte populations were incubated for 18 hours in the presence of 30 nM TSA. The primer extension products are shown with arrows and include the endogenous H4 message as an mRNA preparation control.

Mentions: In order to confirm that we were observing a chromatin mediated effect and to exclude any direct effect of the (CGG)n repeats upon RNA polymerase II transcription, we performed an in vitro "run off" transcription reaction in Hela cell extracts, using primer extension to quantify the mRNA levels. Although these extracts contain the necessary components for mature chromatin formation, they are unable to chromatinise the templates during the short time course of this experiment. Hence any contribution of chromatin to transcriptional repression is removed. Figure 4a shows the primer extension products from such an experiment visualised after separation on a denaturing polyacrylamide gel. In this experiment, the control CMV-CAT reporter construct gives a much greater signal compared to the experimental pHSVtk-CAT DNAs. Relative transcriptional activity of each of the linearised (CGG)n containing reporter DNA's is shown in Figure 4a and shown graphically in Figure 4b. As can be seen, equivalent transcriptional activity is exhibited by all reporter constructs, regardless of repeat number. Thus, in the absence of chromatin formation, we see no (CGG)n induced transcriptional repression. This confirms that we only see transcriptional repression after chromatin formation.


Fragile X (CGG)n repeats induce a transcriptional repression in cis upon a linked promoter: evidence for a chromatin mediated effect.

Chandler SP, Kansagra P, Hirst MC - BMC Mol. Biol. (2003)

The Role of Chromatin in Repeat Induced Transcriptional Repression (a) Primer extension products are shown from run-off RNA transcripts performed in vitro using a Hela cell nuclear extract incubated with pHSVtkCAT, in vitro CpG methylated pHSVtkCAT, pHSVtkCAT-(CGG)27, pHSVtkCAT-(CGG)70 and pHSVtkCAT-(CGG)105 DNAs. All reactions included control of pCMV-CAT DNA. (b) A graphical representation of the detectable transcript in the Helascribe reaction. The transcription was normalised to CMV-CAT as an internal control. (c) Primer extension analysis of the pHSVtkCAT-(CGG)140 reporter and pCMV-CAT injection control. Injections were of 5 ng reporter and 0.3 ng injection control per oocyte. The injected oocyte populations were incubated for 18 hours in the presence of 30 nM TSA. The primer extension products are shown with arrows and include the endogenous H4 message as an mRNA preparation control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153536&req=5

Figure 4: The Role of Chromatin in Repeat Induced Transcriptional Repression (a) Primer extension products are shown from run-off RNA transcripts performed in vitro using a Hela cell nuclear extract incubated with pHSVtkCAT, in vitro CpG methylated pHSVtkCAT, pHSVtkCAT-(CGG)27, pHSVtkCAT-(CGG)70 and pHSVtkCAT-(CGG)105 DNAs. All reactions included control of pCMV-CAT DNA. (b) A graphical representation of the detectable transcript in the Helascribe reaction. The transcription was normalised to CMV-CAT as an internal control. (c) Primer extension analysis of the pHSVtkCAT-(CGG)140 reporter and pCMV-CAT injection control. Injections were of 5 ng reporter and 0.3 ng injection control per oocyte. The injected oocyte populations were incubated for 18 hours in the presence of 30 nM TSA. The primer extension products are shown with arrows and include the endogenous H4 message as an mRNA preparation control.
Mentions: In order to confirm that we were observing a chromatin mediated effect and to exclude any direct effect of the (CGG)n repeats upon RNA polymerase II transcription, we performed an in vitro "run off" transcription reaction in Hela cell extracts, using primer extension to quantify the mRNA levels. Although these extracts contain the necessary components for mature chromatin formation, they are unable to chromatinise the templates during the short time course of this experiment. Hence any contribution of chromatin to transcriptional repression is removed. Figure 4a shows the primer extension products from such an experiment visualised after separation on a denaturing polyacrylamide gel. In this experiment, the control CMV-CAT reporter construct gives a much greater signal compared to the experimental pHSVtk-CAT DNAs. Relative transcriptional activity of each of the linearised (CGG)n containing reporter DNA's is shown in Figure 4a and shown graphically in Figure 4b. As can be seen, equivalent transcriptional activity is exhibited by all reporter constructs, regardless of repeat number. Thus, in the absence of chromatin formation, we see no (CGG)n induced transcriptional repression. This confirms that we only see transcriptional repression after chromatin formation.

Bottom Line: In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte.We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length.Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sangamo BioSciences, 501 Canal Blvd, Ste A100, Point Richmond Tech Center II, Richmond, CA 94804, USA. schandler@sangamo.com

ABSTRACT

Background: Expansion of an unstable (CGG)n repeat to over 200 triplets within the promoter region of the human FMR1 gene leads to extensive local methylation and transcription silencing, resulting in the loss of FMRP protein and the development of the clinical features of fragile X syndrome. The causative link between (CGG)n expansion, methylation and gene silencing is unknown, although gene silencing is associated with extensive changes to local chromatin architecture.

Results: In order to determine the direct effects of increased repeat length on gene transcription in a chromatin context, we have examined the influence of FMR1 (CGG)n repeats upon transcription from the HSV thymidine kinase promoter in the Xenopus laevis oocyte. We observe a reduction in mRNA production directly associated with increasing repeat length, with a 90% reduction in mRNA production from arrays over 100 repeats in length. Using a kinetic approach, we show that this transcriptional repression is concomitant with chromatin maturation and, using in vitro transcription, we show that chromatin formation is a fundamental part of the repressive pathway mediated by (CGG)n repeats. Using Trichostatin A, a histone deacetylase inhibitor, we show reactivation of the silenced promoter.

Conclusions: Thus, isolated fragile X associated (CGG)n repeat arrays can exert a modifying and transcriptionally repressive influence over adjacent promoters and this repressive phenomenon is, in part, mediated by histone deacetylation.

Show MeSH
Related in: MedlinePlus