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Originally published In Press as doi:10.1074/jbc.M413239200 on December 1, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4498-4503, February 11, 2005
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Effect of CAT or AGG Interruptions and CpG Methylation on Nucleosome Assembly upon Trinucleotide Repeats on Spinocerebellar Ataxia, Type 1 and Fragile X Syndrome*{boxs}

David J. Mulvihill{ddagger}§, Kerrie Nichol Edamura§, Katharine A. Hagerman¶||**, Christopher E. Pearson¶||{ddagger}{ddagger}, and Yuh-Hwa Wang{ddagger}§§

From the {ddagger}Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, Program of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and ||Department of Molecular and Medical Genetics, University of Toronto, Ontario M5G 1X8, Canada

Received for publication, November 23, 2004 , and in revised form, December 1, 2004. , and in revised form, December 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleosome packaging regulates many aspects of DNA metabolism and is thought to mediate genetic instability and transcription of expanded trinucleotide repeats. Both instability and transcription are sensitive to repeat length, tract purity, and CpG methylation. CAT or AGG interruptions within the (CAG)n or (CGG)n tracts of spinocerebellar ataxia, type 1 or fragile X syndrome, respectively, confer increased genetic stability to the repeats. We report the formation of nucleosomes on sequences containing pure and interrupted (CAG)n and (CGG)n repeats having lengths above and below the genetic stability thresholds. Increased lengths of pure repeats led to increased and decreased propensities for nucleosome assembly on the (CAG)n and (CGG)n repeats, respectively. CpG methylation of the CGG repeat further reduced assembly. CAT interruptions in (CAG)n tracts decreased nucleosome assembly. In contrast, AGG interruptions in (CGG)n tracts did not affect assembly by hypoacetylated histones. The latter observation was unaltered by CpG methylation of the repeats. However, nucleosome assembly by hyperacetylated histones on interrupted CGG tracts was increased relative to pure tracts and this effect was abolished by CpG methylation. Thus, CAT or AGG interruptions can modulate the ability of (CAG)n and (CGG) tracts to assemble into chromatin and the effect of the AGG interruptions is dependent upon both the methylation status of the DNA and the acetylation status of the histones. Compared with the genetically unstable pure repeats, both interruptions permit a propensity of nucleosome assembly closer to that of random (genetically stable) sequences, suggesting an association of nucleosome assembly of trinucleotide repeats and genetic instability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The expansion of repeat tracts is associated with at least 42 human diseases (1). Although the instability of CAG/CTG repeats is the cause of 12 diseases, six rare chromosomal fragile sites, three of which are disease-associated, are caused by unstable CGG/CCG tracts. In addition to genetic instability, both CAG- and CGG-expanded repeats have been associated with altered transcription regulation and altered chromatin packaging (2, 3). Genetic instability and transcription of the expanded repeats are modulated by various factors including repeat tract length, repeat tract purity, and aberrant CpG methylation. Mechanistically, the effects of each DNA variable may be transmitted by alterations in histone protein-DNA assembly. However, the interrelation of repeat purity upon nucleosome packaging is unknown.

Repeat instability is dependent upon both the length and purity of the repeat tract. Longer alleles are more likely to undergo further expansion than their predecessor alleles.

Sequence interruptions within the repeat tracts confer increased genetic stability to the tracts (4–8). In non-affected individuals, the (CAG)n tract of spinocerebellar ataxia, type 1 (SCA1)1 contains 1–3 CAT interruptions, usually centrally located and separated by one CAG (4, 5). In the normal population, the (CGG)n tract of fragile X syndrome (FRAXA) contains 1–3 AGG interruptions, typically at every tenth repeat (6–8). Longer lengths of the pure repeats correlate with instability and disease. The stability threshold lengths at which increased instability occurs are ≥35 pure CAG and ≥25–34 pure CGG for SCA1 and FRAXA, respectively (4–8). The reasons for the stabilizing effect of the interruptions are not well understood. The limited types of interruptions, CAT and AGG, and the pattern of their placement within the SCA1 and FRAXA repeat tract suggest a degree of specificity in their ability to protect from instability. There have only been a limited number of experimental studies investigating the role of interruptions in repeat instability. Several have used endogenous interruptions and configurations (9, 10), whereas others have used non-typical patterns (11–13). In addition to decreasing the propensity for slipped-strand structure formation (9), facilitating replication fork progression (11), and altering repair of interrupted repeat tracts (13), it has been hypothesized that interruptions may provide genetic stability to the repeat tracts through altered nucleosome conformations (14).

Alterations in nucleosome formation can affect various processes including transcription. Expanded CTG/CAG and CGG/CCG repeat tracts have variously been associated with decreased (15, 16), increased (17, 18), or unaltered (19) transcription rates of the associated or proximal genes. A loss of FMR1 transcription is linked to full CGG expansions (>200 repeats) and aberrant CpG methylation of the repeat and proximal regions (20). Interestingly, "premutation" lengths of FRAXA (CGG)n where n = 40–200 displayed increased levels of transcription compared with shorter non-disease-associated lengths of 30 repeats (17, 18, 21, 22). These expanded lengths were also associated with variant sites of transcription initiation (23). Moreover, these premutation lengths were associated with premature ovarian failure (24) and the recently described fragile X tremor ataxia syndrome (25) that are clinically distinct from FRAXA mental retardation. Altered transcription regulation of genes with expanded repeats is thought to be mediated by altered chromatin packaging at the expanded repeat.

Aberrant CpG methylation is associated with CGG instability in addition to affecting FMR1 transcription. In the absence of aberrant CpG methylation, CGG deletions can occur in the male germ line during development of a FRAXA fetus (26), whereas the expanded and methylated repeats are postnatally stable. Similarly, the absence of CpG methylation may enhance CGG deletions in the somatic tissues of high functioning FRAXA males that inherited an expanded CGG tract that had experienced only partial aberrant CpG methylation (27, 28) There is some suggestion that the effects of CpG methylation on instability are mediated through replication (29).2

Modulation of CpG methylation and histone modifications can affect cytogenetic fragile site expression and transcriptional activity at the FRAXA locus. Cytogenetic fra(X)(q27.3) fragile site expression, which is unique to FRAXA patient cells with CGG expansions, can be blocked by histone acetylation/methylation by exposure to butyrate (30). De-methylation of DNA by 5-azadeoxycytidine can reactivate suppressed FMR1 transcription in FRAXA patient cells (31, 32). De-methylation is coincident with re-association of acetylated histones at the FMR1 locus (33, 34).

In this paper, we examined the effect of repeat interruptions, DNA methylation, and histone acetylation status on the formation of nucleosomes on SCA1 and FRAXA repeats with repeat lengths above and below the genetic stability thresholds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA—Genomic clones containing human SCA1 (pAG49 and pAG44–24B) and FRAXA (pFXA39, pFXA9+27, pFXA53, and pFXA9+9+32) genomic repeat tracts have been constructed to obtain highly purified DNA samples containing repeat lengths and interruption patterns that are present within the human population. The characterization of all of the plasmids has been described in detail (9, 29).

Clones pAG49 and pAG44–24B were digested with restriction enzymes StuI and EagI to yield DNA fragments "49" (305 bp) and "12+1+12+1+14" (290 bp). Plasmids pFXA39, pFXA9+9+9+9, pFXA53, and pFXA9+9+32 were digested with restriction enzymes MaeIII and NheI to yield DNA fragments "39" (206 bp), "9+9+9+9" (206 bp), "53" (248 bp), and "9+9+32" (245 bp), respectively. Fragment "49" contains a pure (CAG)49 tract, whereas "12+1+12+1+14" contains 44 CAG repeats interrupted by four CAT interspersions (Fig. 1). Similarly, fragments "39" and "53" contain pure CGG repeats (39 and 53 repeats, respectively), whereas fragments "9+9+9+9" and "9+9+32" contain CGG repeats interrupted by AGG interspersions (39 repeats interrupted by 3 AGG sequences and 52 repeats interrupted by 2 AGG sequences, respectively) (Fig. 1). Each of the SCA1 and FRAXA genomic fragments contains human flanking sequences. For SCA1, these sequences are 62 nt upstream and 96 nt downstream of the CAG tract (sites 1498–1559 and 1647–1742 from GenBankTM accession number NM000332), and for FRAXA, these sequences are 37 nt upstream and 49 nt downstream of the CGG tract (sites 24–60 and 151–199 from GenBankTM accession number S65791 [GenBank] ). The purified DNAs were radioactively labeled with Klenow fragment of DNA polymerase I (New England Biolabs Inc.) in the presence of [{alpha}-32P]ATP (Amersham Biosciences). To produce methylated DNAs, the above fragments were methylated with SssI methylase (New England Biolabs Inc.) in the presence of S-adenosylmethionine. The degree of methylation was determined by resistance to cleavage with a methylation-sensitive restriction endonuclease HhaI. For use as a control, a 262-bp PCR product was generated from nucleotides 239 to 500 of the pUC19 vector.



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  		FIG. 1.
SCA1 and FRAXA genomic fragments. The fragments used herein were derived from human genomic clones and contained both human non-repetitive flanking sequences (for details see "Materials and Methods") and repeat tracts that were pure or harbored interruptions in the schematically indicated configurations. In the schematic, the CAG and CGG repeats are indicated by hollow circles, whereas the CAT and AGG interrupts are indicated by filled circles. In the fragment nomenclature, the CAT and AGG interruptions are indicated as + signs and the intervening number is that of the contiguous number of CAG or CGG repeats. The length of the human flanking sequences and the total fragment sizes are indicated.

 
Competitive Nucleosome Reconstitution—Hypoacetylated histone octamers were isolated from HeLa cells (35). Also, HeLa cells were treated with 10 mM sodium butyrate for 24 h to generate hyperacetylated histone octamers. Hyperacetylation of core histone proteins was ~33-fold higher compared with hypoacetylated octamers as quantified by Western blot analysis using an antibody to acetylated histone H3 (Upstate Biotechnology).

The ability of DNA fragments to form nucleosomes was assayed using competitive nucleosome reconstitution (36). DNA fragments (50 ng) labeled with 32P were incubated with 2.5 µg of either hypoacetylated or hyperacetylated histone octamers along with 10 µg of calf thymus DNA in a solution containing 2 M NaCl. NaCl was incrementally reduced to a final concentration of 0.1 M. The assembly mixtures were then directly electrophoresed on 5% polyacrylamide gels at 150 volts for 4 h at room temperature to separate free DNA from the nucleosome-assembled DNA. Results were visualized by autoradiography. The amount of DNA in each band was determined by PhosphorImager scanning (Amersham Biosciences). Each DNA was reconstituted in three separate but identical experiments for each set of histone octamers. For each DNA sample, a "no histones" assembly reaction was performed to serve as a control in which the reaction mixture did not contain histone octamers and the DNA was treated in the same manner, including stepwise salt dilution.

The assembling efficiency was represented by the ratio of DNA in complex over free DNA for each sample. The assembling efficiency for the pUC19 fragment was assigned a value of 1. The free energy of nucleosome assembly was calculated according to the equation, E (the repeat-containing fragment) = RT ln(ratio of DNA in complex to free DNA for pUC19 fragment) – RT ln(ratio of DNA in complex to free DNA for the repeat-containing fragment) (37–39). The free energy for the pUC19 DNA was defined as zero. The values are derived from at least three separate experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CAT Interruptions Hinder Hypoacetylated Nucleosome Formation on CAG Tracts—The effect of repeat interruptions on nucleosome assembly was determined by comparing a genomic SCA1 CAG tract containing 44 repeats (multiply) interrupted by 4 CAT units (designated as "12+1+12+1+14," where + signs represent CAT interruptions) with a similar length of 49 pure uninterrupted CAG repeats (Fig. 1). Using hypoacetylated histone octamers, nucleosomes were assembled upon DNA fragments with the interrupted and pure repeats and compared with a random-sequence control pUC19 fragment, representing a "base line" for nucleosome assembly (see "Materials and Methods"). Following gel electrophoresis, the nucleosome-assembled DNAs appeared as retarded bands (Fig. 2A, lanes 1–3). Assembly was quantified as the ratio of the nucleosome-assembled DNA to free DNA and normalized to the unit ratio generated from pUC19 nucleosome assembly (Fig. 2B). Both the pure 49 repeat and the interrupted "12+1+12+1+14" repeat assembled nucleosomes stronger than the control fragment by as much as 16.3 ± 3.9- and 7.9 ± 3.5-fold, respectively. However, nucleosome assembly on the interrupted repeat was significantly reduced by ~2-fold as that for the pure repeat (p = 0.007). Thus, the presence of four CAT interruptions within a tract of 44 CAG repeats can significantly reduce the ability of an expanded repeat to assemble into nucleosomes. However, although reduced, the propensity of the interrupted tract to form nucleosomes was still greater than that of the non-repetitive control, indicating that interruptions reduce but do not abolish the tendency of CTG/CAG to assemble into chromatin.



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  		FIG. 2.
Competitive nucleosome reconstitution of SCA1 CAG-repeating DNAs with and without CAT interruptions. A, autoradiogram of a competitive nucleosome reconstitution experiment. Lanes 1–3, DNA fragments "12+1+12+1+14," "49," and pUC19 assembled with histones. Lanes 4–6, DNA fragments processed in the same way as for the samples in lanes 1–3 but serving as controls without histone octamers. DNA preparation, reconstitution of the fragments with histone octamers, and gel electrophoresis were as described under "Materials and Methods." B, plot of efficiency of nucleosome assembly. The assembling efficiency is represented by the ratio of DNA in complex over free DNA for each sample. The assembling efficiency for the pUC19 fragment was assigned a value of 1. Each DNA was reconstituted in three separate but identical experiments, and the fraction of DNA in the nucleosome-assembled and nucleosome-free DNA bands was measured by a PhosphorImager.

 
AGG Interruptions Do Not Affect Hypoacetylated Nucleosome Formation on Unmethylated or Methylated CGG Tracts—We next determined the effect of AGG interruptions on the formation of hypoacetylated nucleosomes on genomic FRAXA CGG repeat tract by comparing pure repeat tracts having 39 and 53 repeats with similar but interrupted lengths of 39, "9+9+9+9," and 52, "9+9+32," repeats (where + signs represent AGG interruptions) (Fig. 1). Both unmethylated and CpG-methylated ("39M," "9+9+9+9M," "53M," and "9+9+32M") versions of the above DNAs were tested for their ability to assemble nucleosomes. The assembly results of Fig. 3A were quantitated in Fig. 3B. All of the CGG fragments (pure or interrupted, methylated or unmethylated) showed considerably lower abilities to assemble into nucleosomes relative to the control pUC19 fragment reduced to levels of 10–40%. However, unlike CAT-interrupted CAG tracts (above), AGG interruptions in CGG tracts did not significantly affect hypoacetylated nucleosome assembly (pure 53 repeats versus "9+9+32," p = 0.80; pure 39 repeats versus "9+9+9+9," p = 0.10) (Fig. 3). Similarly, interruptions failed to significantly affect hypoacetylated nucleosome formation upon CpG-methylated CGG tracts ("53M" versus "9+9+32M," p = 0.70; "39M" versus "9+9+9+9M," p = 0.97).



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  		FIG. 3.
Competitive nucleosome reconstitution using hypoacetylated histones with FRAXA CGG-repeating DNAs containing AGG interruptions and CpG methylation. A, autoradiogram of a competitive nucleosome reconstitution experiment. Fragments "39" and "53" contain pure CGG repeats (39 and 53 repeats, respectively), whereas fragments "9+9+9+9" and "9+9+32" contain CGG repeats interrupted by three and two AGG interspersions (indicated by + signs), respectively. Methylated DNAs are indicated by M. B, plot of efficiency of nucleosome assembly. Each DNA was reconstituted in three separate but identical experiments, and the assembling efficiency for the pUC19 fragment was assigned a value of 1.

 
CpG Methylation Diminishes Hypoacetylated Nucleosome Formation on Pure and Interrupted CGG Tracts—The effect of DNA methylation on nucleosome assembly over the pure and interrupted CGG repeats was also assessed in Fig. 3. When compared with its unmethylated counterpart, each methylated CGG tract showed a significantly decreased ability to assemble hypoacetylated nucleosomes (pure 53 repeat DNA versus "53M," p = 0.04; "9+9+32" versus "9+9+32M," p = 0.01; pure 39 repeat DNA versus "39M," p = 0.06; "9+9+9+9" versus "9+9+9+9M," p = 0.01). Thus, CpG-methylation strongly decreased nucleosome formation on CGG repeats, regardless of tract repeat purity.

AGG Interruptions Increase Hyperacetylated Nucleosome Formation on Unmethylated CGG Tracts—In vivo, acetylated rather than deacetylated histones are bound to the unmethylated CGG repeat at the FMR-1 gene (34). Therefore, we next determined the effect of AGG interruptions on nucleosome formation using hyperacetylated histone octamers (see "Materials and Methods"). Among the unmethylated DNA samples, a significant increase in hyperacetylated nucleosome formation is exhibited by AGG-interrupted CGG tracts ("9+9+32" versus pure "53" repeat DNA, p = 0.04; "9+9+9+9" versus pure "39" repeat DNA, p = 0.02) (Fig. 4). Also noteworthy is a subtle increased hyperacetylated nucleosome formation on shorter versus longer CGG tracts (compare pure "39" versus pure "53" and "9+9+9+9" versus "9 + 9+32") (Fig. 4). This CGG tract length effect was observed for hyperacetylated but not hypoacetylated histones.



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  		FIG. 4.
Competitive nucleosome reconstitution of FRAXA DNAs in the presence of hyperacetylated histones. A, autoradiogram of a competitive nucleosome reconstitution experiment with the same set of DNA fragments as in Fig. 2. Hyperacetylated histone octamers were isolated from HeLa cells treated with sodium butyrate. B, plot of efficiency of nucleosome assembly. Each DNA was reconstituted in three separate but identical experiments.

 
CpG Methylation Abolishes Increased Hyperacetylated Nucleosome Formation on AGG-interrupted CGG Tracts—Our results noted above indicate a significant increase in hyperacetylated nucleosome formation on CGG repeats containing AGG interruptions as compared with pure CGG repeats. We next determined whether methylating the CGG repeats would alter their ability to assemble into hyperacetylated nucleosomes. Strikingly, the enhanced nucleosome assembly by the AGG-interrupted tracts was abolished by CpG-methylation (Fig. 4, "9+9+32M" versus "53M," p = 0.16; "9+9+9+9M" versus "39M," p = 0.93). This indicates that the improved nucleosome formation mediated by AGG interruptions is dependent upon both the acetylation state of the histones and upon the CpG-methylation state of the DNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, extending our original observation that expanded CTG/CAG and CGG/CCG repeats preferentially assemble (2, 36) or exclude (38, 39) nucleosome formation, we assessed the effect of repeat purity on the ability of these disease-associated repeats to assemble nucleosomes. Specifically, we show that CAT interruptions in SCA1 CAG tracts decreased their preferential nucleosome assembly, whereas AGG interruptions in FRAXA CGG tracts increased nucleosome assembly by hyperacetylated histones, which lessen the nucleosome exclusion ability of pure CGG repeats. Therefore, the common trend of repeat interruptions is to diminish the strong nucleosome-enhanced or -excluded formations, thereby modulating the repeats to be more like random sequences in their ability to assemble into chromatin (Fig. 5). Such effects on chromatin structure may permit the interrupted repeats to be metabolized (with regard to transcription or mutation) more like random sequences rather than like genetically unstable sequences.



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  		FIG. 5.
Comparison of free energies of nucleosome assembly from pure and interrupted CAG- and CGG-repeating DNAs. The trend of repeat interruptions (shown by arrows) is to diminish the strong enhanced-(CAG repeats) or excluded-(CGG repeats) nucleosome formations, thereby modulating the repeats to be more like random sequences in their ability to assemble into chromatin. Data from Fig. 1 for the CAG DNAs and Fig. 3 for the CGG DNAs were used to calculate relative free energy of nucleosome assembly, which is described under "Materials and Methods." The free energy for the pUC19 DNA was defined as zero. The values for all of the DNA fragments are listed in Supplemental Table I.

 
The effect of AGG interruptions on nucleosome assembly of the CGG repeats has been modeled using DNA flexibility as an indicator (14). Flexible dinucleotides GA and AG (part of AGG interruptions) in the sequence cause lower free energy of bending of the CGG sequence and should promote nucleosome assembly. Similarly, AG steps are lost when CAT interruptions are present within CAG tracts. These losses may reduce the enhanced ability to assemble nucleosomes. Interestingly, the short tracts of (GGA)n repeats, which contain the AGG unit, have been shown to be flexible (40) and preferentially assemble into nucleosomes (41). The differences in the ability of repeat tracts to assemble nucleosomes in the presence or absence of interruptions may be intensified for larger expansions. Such differences could affect higher order chromatin organization, which could have biological effects. Indeed, the strong correlation between the unstable chromatin phenotype of the (CCGNN)48 sequence in yeast (42) and the unfavorable energies of nucleosome formation of repeating CCGNN DNA (37) measured in vitro by the same assay used in this study further argues that the propensity of nucleosome assembly measured in vitro can provide insight into chromatin stability in vivo.

Interruptions have been shown to reduce genetic instability of CAG and CGG repeats in humans. In bacterial and SV40 cell systems, the SCA1 and FRAXA clones containing interrupted repeat tracts are more stable than clones containing pure repeats of similar length (29).3 Our data show the ability of interruptions to amend the nucleosome assembly of both repeats from opposite strengths to a level closer to that of non-repetitive genetically stable sequences, suggesting a possible mechanism by which interruption-altered nucleosome formation along trinucleotide repeats could modulate either transcriptional activity or genetic stability of these sequences (Fig. 5). Also, both AGG interruptions and CpG methylation provided genetic stability of CGG tracts in both bacterial and primate (SV40) DNA replication model system.3 Whereas the overall deletion frequencies were similar with methylated or unmethylated DNA, in both systems, the ability to retain AGG interruptions is stronger only with unmethylated DNA in the primate cell system, which correlates with the increased nucleosome assembly of unmethylated, interrupted DNA by hyperacetylated histones. We propose that the stabilization of AGG interruptions and the increased nucleosome-assembling ability by unmethylated, interrupted (CGG)n tracts may be related.

FMR1 gene expression has been shown to be significantly affected by chromatin structure. CGG repeats, in general, display a strong tendency to exclude nucleosome assembly (38, 39, 43). Here we have confirmed this same phenomenon and extended it to include CGG tracts that are CpG-methylated or AGG-interrupted. Alterations in nucleosome assembly mediated by CGG expansions, CpG methylation, and AGG interruptions may modulate chromatin accessibility. Transcription factors USF1, USF2, and {alpha}-Pal/Nrf-1 have been shown to interact with the FMR1 promoter (44, 45). The binding of such proteins to the CGG repeat and the adjacent promoter can influence directly and indirectly the susceptibility of repeat mutation and/or transcriptional levels or transcription start sites of the FMR1 gene. Our study shows that AGG-interrupted CGG tracts assemble hyperacetylated nucleosomes more efficiently compared with pure repeats, which may suggest that the increased transcription of premutation FRAXA repeat lengths (17, 18, 22) may differ for interrupted alleles compared with the pure CGG tracts. Such effects of AGG interruptions may influence the susceptibility of the individual to fragile X tremor ataxia syndrome and/or premature ovarian failure, both of which are associated with premutation CGG lengths. Transcription of expanded CGG repeats in transgenic mice showed intranuclear inclusions similar to fragile X tremor ataxia syndrome patients (46), whereas significant neurodegeneration was observed in transgenic Drosophila expressing expanded CGG repeats (47). Interruptions did not significantly affect poor nucleosome assembly upon CpG-methylated DNAs, which may reflect the dominant inhibitory effect of CpG-methylation on FMR1 transcriptional activity. The altered nucleosome assembly upon AGG- and CAT-interrupted tracts of FRAXA and SCA1 repeats may explain their ability to genetically stabilize the repeats and may also correlate with altered transcription levels.


   FOOTNOTES
 
* This work was supported by grants from the Canadian Institutes of Health Research and the Fragile X Research Foundation of Canada (to C. E. P.) and from the National Institutes of Health (CA85826) (to Y.-H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table 1. Back

§ Both authors have contributed equally to this work. Back

** Supported by National Science Engineering Research Canada. Back

{ddagger}{ddagger} A CHIR Scholar and a Canadian Genetic Disease Network Scholar. Back

§§ To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5050; Fax: 732-235-3232; E-mail: wangyu{at}umdnj.edu.

1 The abbreviations used are: SCA1, spinocerebellar ataxia, type 1; FRAXA, fragile X syndrome; nt, nucleotide. Back

2 K. N. Edamura, M. Leonard, and C. E. Pearson, unpublished data. Back

3 K. N. Edamura and C. E. Pearson, unpublished data. Back



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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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