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BMC Mol Biol
2003 Mar 21;4:3. doi: 10.1186/1471-2199-4-3.
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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
.
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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.
Figure 1. FMR1 (CGG)n TK-CAT constructs. (a) A schematic representation of the constitutive HSVtk-CAT promoter showing the position of the (CGG)n repeat cloning site. The distance from the start site of transcription to the repeats, in base pairs, is similar as that found in the native FMR1 promoter. The arrow represents the position of the primer used for the analysis of transcripts by primer extension and the thickened line represents pBR322 vector-derived DNA. (b) An agarose gel showing reporter plasmid digests in which HindIII treatment releases the (CGG)n and flanking sequences. Lanes 1, 2 and 3 are digested pBR TK-CAT plasmids containing (CGG)27, (CGG)70 and (CGG)105 repeat lengths respectively. The released fragments are shown by the arrows corresponding to (CGG)105, (CGG)70 and (CGG)27 respectively. The marker lane contains a 1 kb ladder
Figure 2. Increased Transcriptional Repression Correlates with Repeat Length. (a) Primer extension analysis of transcripts from the HSV-TK promoter adjacent to increased lengths of (CGG)n triplet repeat. None: no injected DNA, CAT: 0.3 ng pCMV-CAT alone. All other lanes represent co-injections of 3 ng of reporter DNA and 0.3 ng of control (pCMV-CAT) DNA per ooctye. Lane 1: pHSVtk-CAT vector; Lane 2: methylated pHSVtk-CAT vector. Lanes 3 to 6: pHSVtk-CAT containing 27, 70, 105 and 140 (CGG) triplet repeats, respectively (un-methylated). Post injection, oocytes were incubated for 18 hours at 18°C. Each extension reaction used RNA from 7 oocytes. (b) A graphical representation of the relative transcription from the HSV-TK promoter after normalisation to the co-injected pCMV-CAT control and for background signal. Lane numbers correspond to those in panel A.
Figure 3. Repeat-Induced Transcriptional Repression is Time Dependent. (a) Primer extension products are shown from mRNA pools taken from oocytes injected with 5 ng of pHSVtk-CAT (no repeats), or pHSVtk-CAT (CGG70) and with 0.3 ng pCMV-CAT as a control for the transcriptional competence of the oocytes. Lane numbers refer to time in hours post-injection. CON represents no oocyte injection and CAT refers to an injection of 0.3 ng pCMV-CAT alone. Primer extension products corresponding to, CMV-CAT and HSVtk-CAT mRNAs are shown. (b) A graphical representation of levels of transcription from the HSV-TK promoter based upon data shown in panel A. Transcription levels are normalised to the co-injected pCMV-CAT control and for background signal. (c) A Southern blot of a chloroquine supercoiling assay of injected pHSVtk-CAT (CGG)70 DNA. Time points of injection mirror those of panel (b). Lane I represents input DNA, other lane numbers refer to time in hours post-injection.
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.
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