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Human Molecular Genetics Advance Access originally published online on February 2, 2005
Human Molecular Genetics 2005 14(6):785-797; doi:10.1093/hmg/ddi073
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Human Molecular Genetics, Vol. 14, No. 6 © Oxford University Press 2005; all rights reserved

Homologous pairing of 15q11–13 imprinted domains in brain is developmentally regulated but deficient in Rett and autism samples

Karen N. Thatcher, Sailaja Peddada, Dag H. Yasui and Janine M. LaSalle*

Medical Microbiology and Immunology and Rowe Program in Human Genetics, School of Medicine, University of California, Davis, CA, USA

* To whom correspondence should be addressed at: Medical Microbiology and Immunology, One Shields Avenue, Davis, CA 95616, USA. Tel: +1 5307547598; Fax: +1 5307528692; Email: jmlasalle{at}ucdavis.edu

Received December 17, 2004; Accepted January 24, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Rett syndrome (RTT), caused by mutations in MECP2 (encoding methyl CpG binding protein 2), and Angelman syndrome (AS), caused by maternal deficiency of chromosome 15q11–13, are autism-spectrum neurodevelopmental disorders. MeCP2 is a transcriptional repressor of methylated genes, but MECP2 mutation does not directly affect the imprinted expression of genes within 15q11–13. We tested a potential role for MeCP2 in the homologous pairing of imprinted 15q11–13 alleles in human brain tissue and differentiated neurons by fluorescence in situ hybridization (FISH). FISH analysis of control cerebral samples demonstrated a significant increase in homologous pairing specific to chromosome 15 from infant to juvenile brain samples. Significant and specific deficiencies in the percentage of paired chromosome 15 alleles were observed in RTT, AS and autism brain samples when compared with normal controls. SH-SY5Y neuroblastoma cells also showed a significant and specific increase in the percentage of chromosome 15q11–13 paired alleles following induced differentiation in vitro. Transfection with a methylated oligonucleotide decoy specifically blocked binding of MeCP2 to the SNURF/SNRPN promoter within 15q11–13 and significantly lowered the percentage of paired 15q11–13 alleles in SH-SY5Y cells. These combined results suggest a role for MeCP2 in chromosome organization in the developing brain and provide a potential mechanistic association between several related neurodevelopmental disorders.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Autism is a complex genetic disorder involving multiple chromosomal loci and environmental influences (1Go,2Go). Several autism-spectrum neurodevelopmental disorders with known genetic causes can be useful in understanding the multiple genetic and epigenetic pathways involved in the etiology of autism. Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in females caused by mutation in MECP2 on Xq28 (3Go). Similar to autism, onset of symptoms are delayed until 6–18 months of age and include severe mental retardation with absence of speech, stereotypic hand movements and epileptic seizures. Both autism and RTT fall under the heading of pervasive developmental disorders (PDD). Although mutations have been found in a few cases of autism, several large studies have ruled out mutations in the coding region of MECP2 as a significant genetic cause of autism (4Go–8Go). Angelman syndrome (AS) is clinically very similar to RTT (except for the lack of regression in skills) and severe mental retardation, epilepsy and autistic features combine with a happy disposition and inappropriate laughter (9Go). AS is caused by maternal deficiency of chromosome 15q11–13 by maternal deletion, paternal disomy, maternal UBE3A mutation or maternal methylation defects. Prader–Willi syndrome (PWS) is a distinct neurodevelopmental disorder caused by paternal deficiency of 15q11–13. In one study, ~2% of AS patients had mutations in MECP2 (10Go), and another recent study found 15q11–13 rearrangements in 5% of RTT patients (11Go). Interestingly, 1–2% of autism patients have been described with maternal duplications of 15q11–13 by interstitial duplications or marker chromosomes (12Go). In addition, several recent studies have demonstrated linkage of autism to polymorphisms within 15q11–13 near UBE3A (13Go), GABRB3 (14Go) and GABRG3 (15Go). We have recently demonstrated deficiencies in MeCP2 expression in autism, AS and PWS postmortem brain samples arranged on a tissue microarray (16Go). Furthermore, UBE3A and GABRB3 expression defects were observed in RTT and autism brain samples and Mecp2-deficient mouse brain (17Go). Together, these observations suggest phenotypic and genetic overlap among autism, RTT and AS.

MECP2 encodes methyl CpG-binding protein 2 that binds to methylated CpG sites within nuclear heterochromatin (18Go,19Go) and is predicted to be a transcriptional repressor of methylated genes through its interaction with molecules such as Sin3A, histone deacetlyase (HDAC) (20Go), DNA methyltransferase (DNMT1) (21Go) and histone methyltransferase (22Go). As the 15q11–13 locus is subject to parental imprinting and characterized by allele-specific methylation and transcription, the first hypothesis was that MeCP2 was essential for the repression of the methylated imprinted genes within 15q11–13. We have previously disproven this simple hypothesis, however, by demonstrating that several imprinted genes within 15q11–13 and 11p15 retained monoallelic expression in MECP2-mutant lymphocyte clones and Rett brain samples (23Go) as well as Mecp2-deficient mouse brain (17Go). MeCP2 is most highly expressed in the nuclei of large mature neurons within the CNS (24Go–26Go). Because changes in nuclear heterochromatin and chromosome positions accompany activation of neurons (27Go), we now investigated whether MeCP2 may be involved in the organization of chromosomes within neuronal nuclei and thus have a more indirect effect on gene expression within 15q11–13.

Specific organization of homologous chromosomes has previously been observed in three-dimensional reconstruction studies of neuronal nuclei, with 9q12 and 1q12 showing association around the nucleolus (28Go,29Go). In other studies on human brain, chromosome 1 and 17 showed evidence for somatic pairing (30Go,31Go). In addition, dynamic changes in the position and clustering of centromeres in Purkinje neurons occur during early postnatal development, associated with the nucleolus (32Go). Homologous association of 15q11–13 domains has been previously observed during late S-phase in lymphocytes (33Go) but has not been previously examined in neurons or brain tissue. In this report, we demonstrate evidence for significant increased homologous pairing of 15q11–13 domains during normal postnatal brain development in human brain. In addition, we demonstrate that brain samples from several related neurodevelopmental disorders show deficiencies in homologous pairing specifically for 15q11–13. We further implicate MeCP2 in the mechanism of homologous pairing by specifically blocking its binding to endogenous chromatin and demonstrating a significant reduction in homologous pairing of 15q11–13 domains in cultured neuroblastoma cells. These results open up many new areas of investigation for understanding the roles of MeCP2 and 15q11–13 during normal neuronal maturation and the pathogenesis of several neurodevelopmental disorders.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Developmental changes in homologous pairing of 15q11–13 domains
In order to investigate the possibility that homologous association of imprinted 15q11–13 domains may occur in brain during normal development, we devised a fluorescence in situ hybridization (FISH) protocol for detection of chromosome 15 in sections from human postmortem brain. A probe specific to the pericentromeric region of chromosome 15 (CEP 15-SpectrumGreen, or D15Z1) was used as it is physically close to 15q11–13 and previously showed significant transient association in cycling lymphocytes (33Go). As a control, another pericentromeric probe specific for chromosome 11 (CEP 11-SpectrumOrange) was used that did not show homologous pairing in brain. In addition, a probe for a single copy locus within 15q11–13 (GABRB3-SpectrumOrange, ~5 Mb from CEP15) was chosen to directly examine 15q11–13 pairing. A control single copy probe was chosen from another acrocentric chromosome (LSI 22-SpectrumGreen, from BCR-ABL on chromosome 22, also ~5 Mb from the pericentromeric heterochromatin), because acrocentric effects of nuclear organization were expected.

In order to accurately compare FISH signals from multiple human brain samples and control for slide-to-slide variability, we used a tissue microarray approach (34Go,35Go). Sections from a previously described tissue microarray containing triplicate 600 µm cores of frontal cortex samples from 28 different controls and patients with neurodevelopmental disorders (16Go) were hybridized with the FISH probe combinations described earlier and then counterstained with DAPI (blue fluorescence). Representative images are shown in Figure 1A and B. Because tissue sections can result in incomplete nuclei that may be missing FISH signals, three different possible FISH patterns were scored per nucleus and are shown to the right of the graph in Figure 2. Nuclei with only one FISH signal (‘one spot’) could be the result of either a missing FISH signal due to sectioning or two overlapping FISH signals due to pairing of alleles (Fig. 1B). In contrast, ‘paired’ nuclei were those scored as having two closely spaced but discernable FISH spots per nucleus, whereas ‘unpaired’ nuclei showed two FISH signals per nucleus >2 µm apart, based on the threshold set in a previous study taking actual distance measurements (33Go). The percentage of nuclei (mean±SEM) for each of the three scoring categories is graphed in Figure 2. A significant increase was observed in the percentage of ‘one spot’ nuclei (white bars) in juvenile/adult when compared with infant control samples for CEP 15, but not CEP 11, suggesting a specific change in the organization of chromosome 15 not likely due simply to lost FISH spots. In addition, a specific and significant increase in ‘one spot’ nuclei was observed for the 15q11–13-specific GABRB3 probe but not for the control LSI 22 probe in juvenile/adult when compared with infant brain samples. A larger number of ‘paired’ nuclei were observed for LSI 22 than for CEP 11 control probes, demonstrating an expected effect on organization of acrocentric chromosomes around the nucleolus (36Go). A significant developmental-specific change in paired alleles was not observed with LSI 22, however, suggesting that the developmental increase in ‘one spot’ nuclei observed with GABRB3 was not simply due to changes in acrocentric organization. These combined results suggest a specific pairing of 15q11–13 alleles during postnatal brain development that correlates with the timing of increased MeCP2 expression (24Go,37Go).



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  		Figure 1. Representative images of FISH signals obtained for these studies from human postmortem brain samples (A, B) or SH-SY5Y neuroblastoma cell lines (C–F). (A) A section of cerebral cortex from a 1-month-old infant brain hybridized with probes specific for pericentromeric regions of chromosome 15 (CEP 15, green) and chromosome 11 (CEP 11, red). (B) The same probe combination as in (A) hybridized to a 2.5-year-old juvenile cerebral section showing an increase in the number of nuclei showing one green spot while still maintaining two red spots (yellow arrows). Nuclei were counterstained with DAPI (blue). Partial nuclei without detectable FISH spots were not scored. (C) SH-SY5Y neuroblastoma cells prior to differentiation show growth in clusters without long axonal projections. Hybridization with a CEP 15 (green) and a 15q11–13 specific probe (GABRB3, red) resulted in the majority of nuclei (blue) showing two unpaired spots of each color. (D) Following PMA-induced differentiation for 3 days, SH-SY5Y cells show long axonal projections (white arrows) and pairing of both CEP 15 (green) and GABRB3 (red) signals. Two different fields are shown because of the lower density of differentiated SH-SY5Y cells in culture. (E) Undifferentiated and (F) 3 day PMA differentiated SH-SY5Y nuclei hybridized with control FISH probes CEP 11 (red) and LSI 22 (green) do not show evidence for increased pairing of signals following differentiation. For images in (C–F), phase contrast images (grayscale) were overlayed on three-color FISH images to demonstrate projections.

 


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  		Figure 2. Specific developmental change in the number of chromosome 15 FISH signals per nucleus in control human brain tissue. A tissue microarray containing frontal cortex samples from 28 different controls and patients with neurodevelopmental disorders has been previously described (16Go). Replicate section of the tissue microarray were each hybridized with either CEP 15 (green) and CEP 11 (red) or LSI 22 (green) and GABRB3 (red) and counterstained with DAPI (blue). Signals of each probe were scored in each nucleus as one of the three patterns shown at right: ‘one spot’ nuclei showed only one spot, ‘paired’ nuclei showed two closely spaced but discernable spots and ‘unpaired’ nuclei showed two distinct spots. Control samples were categorized as infant (1–56 days) or juvenile to adult (2.5–36 years), and the total number of individuals and nuclei scored are shown below the graph. The percentage of nuclei in each of the three categories (mean ± SEM) of different samples and replicate hybridizations are graphed. A significant increase was observed in the percentage of ‘one spot’ nuclei (white bars) following infancy in control samples for CEP 15 and GABRB3, but not CEP 11 or LSI 22. A corresponding significant decrease in the percentage of ‘unpaired’ nuclei (dark gray bars) was observed for both chromosome 15 specific probes, but not control probes. ***P<0.0005 by t-test. Data for scoring of individual brain samples is shown in Supplementary Material, Table S1 and Figure S1. Five additional infant brain samples are also included in Supplementary Material, Figure S1.

 
Defects in homologous pairing of 15q11–13 observed in several neurodevelopmental disorders
The tissue microarray used for the analysis of multiple brain samples in Figure 2 also contained samples of age- and region-matched cerebrum from patients with RTT (n=6), autism (n=5), AS (n=1), PWS (n=2) and PDD (n=1). The nuclei within each sample were scored in an identical fashion to the control samples described previously and the results are graphed in Figure 3. In order to test the significance of changes in the FISH patterns, samples with similar diagnoses were grouped and compared with normal controls (mean 18.1 years). AS and PWS samples were grouped together because of the paucity of samples in these categories and the expected loss of homologous pairing for both disorders (33Go). The most significant changes in FISH patterns from control samples were observed using the GABRB3 15q11–13-specific probe, as both RTT and autism samples showed a significant increase in the percentage of ‘unpaired’ nuclei and a corresponding decrease in the percentage of ‘one spot’ nuclei. The PWS 865 and AS 293 samples graphed for GABRB3 are monosomic for 15q11–13 because of deletions and therefore were expected to show primarily ‘one spot’ nuclei. A PWS uniparental disomy sample (PWS 1290) was also scored and showed defects in GABRB3 pairing (Supplementary Material, Table S1). In contrast, control acrocentric probe LSI 22 showed no significant changes in the percentage of ‘one spot’ or ‘paired’ nuclei, suggesting a specific loss of 15q11–13 pairing in these neurodevelopmental disorder samples. Furthermore, the results obtained with pericentromeric probe CEP 15 showed significant increases in the percentage of ‘unpaired’ alleles in RTT, autism and PWS/AS samples, whereas those with the control CEP 11 probe showed no significant differences. These results suggest defects in homologous pairing of 15q11–13 alleles in several related neurodevelopmental disorders.



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  		Figure 3. Significant differences in the number of chromosome 15 specific FISH signals in brain samples from patients with neurodevelopmental disorders. Brain samples from patients with neurodevelopmental disorders were grouped according to diagnosis and results of the scoring of FISH signals described in Figure 2 were compared with juvenile and adult control samples for significance. The total number of nuclei scored and number of brain samples in each category are shown below the graph. The percentage of nuclei in each of the three categories (mean ± SEM) of different samples and replicate hybridizations is shown. Only samples with >90% hybridization efficiency were scored, resulting in slight differences among the total number of samples scored for each probe. Significant increases in the percentage of ‘unpaired’ nuclei were observed for in all three categories of neurodevelopmental disorders (RTT, autism and PWS/AS) for CEP 15 and GABRB3 (with the exception of PWS/AS) when compared with control samples. Both PWS and AS samples graphed for GABRB3 are monosomic for 15q11–13 and, therefore, served as a control for non-specific hybridization signals (<5%). In contrast to the differences in FISH spot signals observed for chromosome 15 specific probes, no significant differences were observed in the FISH patterns using either of the control probes CEP 11 or LSI 22. The average nuclear area (73.8±2.1 µm2) was not significantly different between different sample categories on the tissue microarray as determined from laser scanning cytometry data collected in previous analyses (17Go). *P<0.05, **P<0.005 and ***P<0.0005 by t-test. Data for scoring of individual brain samples is shown in Supplementary Material, Table S1.

 
Although the result of identifying defects in 15q11–13 organization in patient samples with different genetic mutations was somewhat unexpected, our previous investigation of MeCP2 expression levels on the same tissue microarray identified multiple abnormalities in MeCP2/MECP2 expression in all of the neurodevelopmental disorder samples (16Go). We therefore hypothesized that MeCP2 may play an important or necessary role in the organization of 15q11–13 alleles.

Increased homologous pairing of chromosome 15 following induced differentiation of SH-SY5Y neuroblastoma cells
Because the problem of incomplete nuclei was inherent to use of brain tissue, we sought an independent cell culture system to investigate the changes in the organization of 15q11–13 domains during neuronal differentiation and to directly test the necessity of MeCP2 in the process. SH-SY5Y neuroblastoma cells were selected because they can be induced to undergo differentiation within 3 days using PMA, resulting in a morphologic change in the extension of axonal projections and the increased expression of neuron specific enolase (38Go). In addition, SH-SY5Y cells are diploid for most chromosomes, including those sampled by our FISH probes (39Go). Nuclei showing more than two spots because of replication or aneuploidy were infrequent and excluded from scoring. Representative images are shown in Figure 1D–F, with projections indicated by arrows. MeCP2 expression is significantly increased 24–72 h following PMA treatment (40Go), making it a good model for the developmental maturation stage characteristic of neurons expressing high levels of MeCP2 in the developing brain (37Go).

SH-SY5Y neuroblastoma cells were cultured on glass slides, fixed either before (untreated) or 72 h following differentiation with PMA (PMA treated) and hybridized with the same FISH probes described previously for brain tissue (see representative images in Fig. 1C–F). Because fixation of cells results in whole nuclei, FISH patterns were scored as simply paired (one spot or two spots <2 µm apart) or unpaired. The results, graphed in Figure 4, demonstrate a significant increase in the percentage of paired alleles following PMA-induced differentiation for nuclei hybridized with CEP 15 and GABRB3 probes, but not control CEP 11 or LSI 22 probes. These results provide additional support to the conclusion that chromosome 15q11–13 alleles show increased pairing during neuronal differentiation. As MeCP2 expression is upregulated by 48 h following differentiation in SH-SY5Y cells (40Go) (data not shown), the developmental regulation would coincide with that of the 15q11–13 homologous pairing.



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  		Figure 4. SH-SY5Y neuroblastoma cells show an increase in the percentage of 15q11–13 paired alleles following induced differentiation. SH-SY5Y neuroblastoma cells were cultured on glass slide chambers and fixed before (untreated) or 72 h following differentiation with 16 nM PMA (PMA treated), then hybridized with either CEP 15 (green) and GABRB3 (red) or CEP 11 (red) and LSI 22 (green). Representative images are shown in Figure 1C–F. Nuclei were scored as either ‘unpaired’ or ‘paired’ for simplicity, but one experiment scored for ‘one spots’ is shown in Supplementary Material, Figure S2. The percentage of ‘paired’ nuclei is graphed for untreated (white bars) and PMA treated (gray bars) as the mean±SEM of three to four replicate experiments, each with 100–500 nuclei scored. A significant increase in the percentage of nuclei showing pairing of CEP 15 and GABRB3 was observed following SH-SY5Y differentiation. In contrast, no significant differences were observed using control probes CEP 11 or LSI 22. *P<0.05, by t-test.

 
Transfected methylated decoys for MeCP2 partially block neuronal differentiation and homologous pairing
To directly test the role of MeCP2 in the nuclear organization of 15q11–13 alleles, we sought to temporarily disrupt the function of MeCP2 in the SH-SY5Y system. We chose to use an oligonucleotide decoy approach because of the success of these systems for blocking targets of transcription factors (41Go). Two different double-stranded phosphorothioate oligodeoxynucleotides were obtained from GeneDetect.com: the wild-type MeCP2 binding sequence (MDWT) contained two CpG methylation sites, whereas the mutant sequence (MDMT) was identical except for the replacement of CG with AT and therefore served as a specificity and transfection control. FITC-labeled oligonucleotides showed a high transfection efficiency by quantitative laser scanning cytometry and stability within SH-SY5Y cells for 72 h (data not shown). For the results graphed in Figure 5, SH-SY5Y cells were transfected with MDWT or MDMT 12 h prior to PMA addition, then fixed 72 h later. The analysis of FISH signals from three experimental replicates demonstrated a significant difference between MDWT and MDMT transfected cells in the percentage of nuclei showing paired alleles of GABRB3, but not CEP 15, CEP 11 or LSI 22. These results suggest that MeCP2 has a specific effect on the organization of 15q11–13 alleles but is not involved in the nucleolar organization of CEP 15.



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  		Figure 5. Transfection of methylated MeCP2 decoy into SH-SY5Y cells prior to differentiation significantly reduces the homologous pairing of GABRB3 alleles. SH-SY5Y neuroblastoma cells were cultured on glass slide chambers and transfected with either MDWT (containing two methylated CpGs) or MDMT (without CpGs) oligonucleotide decoys. Twelve hours following transfection, PMA was added to induce differentiation and fixed after 72 h in culture. The percentage of paired nuclei were scored and the results shown are the mean ±SEM of nine replicate scorings of seven replicate hybridizations from four replicate transfections, each with 100–300 nuclei scored. A significant decrease in the percentage of paired alleles of GABRB3 was observed for cells transfected with the methylated MeCP2 decoy (MDWT transfected, light gray bars) when compared with the control (MDMT transfected, dark gray bars). In contrast, no significant difference was observed using CEP 15 or either of the control probes. *P<0.05, by t-test.

 
In order to confirm that the MDWT transfection specifically blocked the binding of MeCP2 to endogenous methylated CpG targets within 15q11–13, we performed chromatin immunoprecipitation (ChIP) on SH-SY5Y cells transfected with MDWT or MDMT and differentiated for 48 h as well as untreated and PMA treated but untransfected controls. Two different antibodies reactive to the C-terminal epitope of MeCP2 were used to IP endogenous fragments bound to MeCP2 and the resulting DNA samples were assayed by PCR. Representative results are shown in Figure 6A for the methylated SNURF/SNRPN promoter within the 15q11–13 imprinting control region (42Go) and an expressed and unmethylated housekeeping control gene (GAPDH). The untransfected controls showed a high recovery of the SNURF/SNRPN promoter region from total input DNA by anti-MeCP2 ChIP with both antibodies. An increase in MeCP2 binding following differentiation was also apparent, concordant with the increase in MeCP2 expression by 48 h (40Go). In contrast, differentiated SH-SY5Y cells transfected with MDWT showed a 10-fold reduction of SNRPN/SNURF containing fragments precipitated with anti-MeCP2 when compared with MDMT transfected controls. As expected, GAPDH sequences were not recovered at a detectable level following ChIP with anti-MeCP2. To confirm the reproducibility of the ChIP results, semi-quantitative PCR results from four separate experiments graphed in Figure 6B show a significant decrease in the amount of SNURF/SNRPN sequences (as a ratio of input DNA) immunoprecipitated from differentiated SH-SY5Y cells transfected with the MDWT decoy when compared with the MDMT control (P<0.005). To determine whether the effect of MDWT transfection was specific to MeCP2 binding, antibodies to all other known methyl binding domain (MBD)-containing proteins (MBD1, MBD2, MBD3 and MBD4) were used on the same chromatin preparations in the ChIP assay. The results of two separate experiments with each antibody are graphed in Figure 6B. MBD1 and MBD2 showed an increase in binding to the SNURF/SNRPN promoter following differentiation and a slight non-specific blocking effect with transfection of both MDWT and MDMT, suggesting a non-methyl-specific effect of the decoy transfection. MBD3 and MBD4 showed a decrease in binding to the SNURF/SNRPN promoter following differentiation and therefore no significant changes due to decoy transfection. These results demonstrate that transfection with the methylated decoy resulted in specific decreased binding of MeCP2 to an endogenous methylated target within the imprinting control region of 15q11–13 that could potentially explain the reduced homologous pairing of this region.



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  		Figure 6. ChIP analysis of MeCP2 bound to the SNURF/SNRPN promoter demonstrates that the methylated decoy specifically blocked binding of MeCP2 to an endogenous site within 15q11–13. (A) Representative gel of PCR products using primers specific for the SNURF/SNRPN promoter following ChIP with two different C-terminal MeCP2 reactive antibodies (Aves chicken or Upstate rabbit). The total DNA isolated from chromatin prior to IP (Input) was used as a positive control. The PCR fragment was highly represented in differentiated SH-SY5Y cells or cells transfected with control decoy (MDMT) but not in chromatin isolated from cells transfected with methylated MeCP2 decoy (MDWT). In contrast, the GAPDH promoter was not detectable following ChIP with anti-MeCP2. Additional PCR experiments performed with or without predigestion with a methyl-sensitive enzyme demonstrated that MeCP2 was bound to the methylated maternal allele, as expected (Supplementary Material, Fig. S3). (B) The mean ± SEM of four separate ChIP experiments using anti-MeCP2 (both antibodies) are graphed as a ratio of the immunoprecipitated to the ‘input’ band intensities. For ChIP performed with anti-MeCP2, the SNURF/SNRPN promoter sequence was significantly lower (P<0.005) in MDWT transfected cells (light gray bars) when compared with MDMT transfected cells (hatched bars), demonstrating that binding of MeCP2 to an endogenous methylated site within 15q11–13 was specifically blocked by the MDWT decoy. Other MBD proteins (mean ± SEM of two replicates each) did not show significant differences between MDWT and MDMT transfected cells in the recovery of the SNURF/SNRPN promoter or were undetectable (no bar) by ChIP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Despite the identification of several candidate genes for neurodevelopmental disorders, the precise role of the gene products in the development or differentiation of neurons remains uncharacterized. MeCP2 has been predicted to be part of an epigenetic pathway of gene expression during neuronal maturation (1Go), but evidence is lacking for a direct role of MeCP2 in silencing imprinted genes within 15q11–13. Maternal 15q11–13 deficiency causes AS, whereas maternal duplication of 15q11–13 is observed in autism, implying that correct parental chromosome dosage is important for normal brain development. Recent reports of trans effects of mutations in imprinted regions (43Go–45Go) prompted us to examine the possibility of homologous interactions of 15q11–13 domains in human brain. In this study, a novel investigation of the organization of homologous 15q11–13 alleles has revealed developmental changes in normal brain development and defects in RTT, AS/PWS and autism. In addition, the novel implication of MeCP2 in the process of homologous 15q11–13 pairing provides an important molecular link between these different neurodevelopmental disorders.

The organization of chromosomes in interphase nuclei has been predicted to be a potentially important mechanism of regulating gene expression during cellular differentiation, especially in neurons (30Go,46Go–48Go). Homologous pairing has been previously observed in human brain for pericentromeric regions of chromosomes 1, 8 and 17 (30Go,31Go,48Go). Homologous associations of human 15q11–13 domains were previously observed in lymphocytes but restricted to the late S-phase of the cell cycle and cells with a biparental contribution of 15q11–13 (33Go). 15q11–13 pairing was also observed at a high frequency in cycling fibroblast and NT2 neuronal cultures (49Go). Homologous pairing of the syntenic region on distal murine chromosome 7 in mouse fibroblasts was also observed exclusively in S-phase (50Go). Our finding of homologous pairing of 15q11–13 domains in postnatal human brain samples containing mostly nuclei in G0 suggests that the association of certain homologous regions can occur both during the cell cycle and during the cellular differentiation. As postmitotic neurons undergo substantial changes in synapse formation and dendritic branching during postnatal development, changes in nuclear organization may reflect dynamic transcriptional patterns.

In addition to the developmental changes in organization of 15q11–13 homologs in human brain, we demonstrate significant defects in homologous pairing in several related neurodevelopmental disorders. The defects in CEP 15 pairing of PWS and AS patients with paternal or maternal deletions of 15q11–13 were expected as this result had been observed previously in lymphocytes (33Go). Maternal duplications of 15q11–13 in 1–2% of autism patients (12Go) also suggest that mismatching of 15q11–13 homologous pairing may cause an autistic phenotype. The novel demonstration of defects in homologous pairing in patient brain samples without detectable cytogenetic abnormalities in 15q11–13, however, suggests that RTT and autism may share defects in an overlapping pathway that regulates homologous pairing of 15q11–13 regions in brain. We have recently demonstrated defects in the level of MeCP2 expression in the same neurodevelopmental samples from the same tissue microarray (16Go). Two of the RTT patients have truncation mutations in MECP2, but the remaining two RTT and all five autism samples have no detectable mutation in MECP2 (including ~1 kb of promoter and exon 1), but significant differences in MeCP2 protein expression (16Go). Interestingly, two samples (PDD 144 and AUT 732) shown to have increased MeCP2 expression compared with controls also showed reduced 15q11–13 pairing (Supplementary Material, Table S1). As higher MeCP2 expression in transgenic mice also causes a severe neurologic phenotype (51Go,52Go), perhaps a precise level of MeCP2 binding is required for nuclear organization of 15q11–13 in brain. On the basis of these combined results, we hypothesized that MeCP2 may be directly involved in the homologous pairing of 15q11–13 domains in the postnatal brain.

The analysis of brain samples has several potential limitations for the analysis of homologous pairing by FISH. First, as FISH is performed on sectioned tissue, not all nuclei are complete and ‘one spots’ may represent absent signals in addition to paired alleles and the developmental changes could simply be due to increased nuclear area. The lack of a significant increase during development in ‘one spots’ of control probes CEP 11 and LSI 22, however, argues against this trivial explanation and suggests a specific homologous pairing of chromosome 15 during postnatal brain development. A second potential problem could be the recently reported aneuploidy of normal postnatal neurons (53Go). Our results are not consistent with monosomy of chromosome 15 being an explanation for our results because there was an increase in the percentage of both ‘paired’ and ‘one spot’ nuclei for CEP 15 during development. The use of an in vitro system for inducing neuronal maturational differentiation and elevated MeCP2 expression was essential for confirming that the changes in homologous pairing of chromosome 15 could be experimentally induced. Our results demonstrate that significant increases in the percentage of nuclei showing chromosome 15 paired alleles is observed within 72 h following differentiation of SH-SY5Y neuroblastoma cells. The GABRB3 probe signals were closer than the CEP 15 or LSI 22 probe signals, as evident from the increased number of ‘one spots’ in these intact nuclei (Supplementary Material, Fig. S2). The FISH experiments, however, do not provide evidence for an actual physical contact between alleles but instead indicate non-random nuclear organization that could come from two alleles sharing a ‘transcription factory’ (54Go) or ‘chromatin hub’ (55Go).

Although MeCP2 acts as a transcriptional repressor of methylated gene constructs (20Go,56Go), a paucity of methylated genes showing increased expression in mutant brain has been identified by genome-wide expression profiling (57Go,58Go). Brain derived neurotrophic factor (BDNF), whose activity-dependent transcriptional activation is regulated by methylation (59Go), shows significantly increased basal transcription in Mecp2-null cultured neurons (60Go). Recently, two genes within an imprinted domain (Dlx5 and Dlx6) have demonstrated to exhibit increased transcription in Mecp2-deficient brain (61Go). MeCP2 has been predicted to have additional roles, including HDAC-independent transcriptional repression (62Go), association with WW-domain splicing factors (63Go), matrix attachment activity (64Go), chromatin compaction activity (65Go) and silent chromatin looping (61Go). In this report, we demonstrate another potential role for MeCP2 in long-range interactions of an imprinted chromosomal region essential for normal brain development. By blocking the binding of MeCP2 by a methylated decoy approach, we demonstrate a significant defect in homologous pairing when compared with the transfection control, suggesting that MeCP2 is involved in the pathway of 15q11–13 allele pairing. ChIP assays confirmed that binding of MeCP2 to the SNURF/SNRPN promoter within 15q11–13 was significantly reduced by the methylated decoy.

Recently, a second isoform of MeCP2 has been described that arises from alternative splicing of exon 2 and results in a change in the N-terminus (66Go,67Go). Because our ChIP experiments utilized C-terminal reactive antibodies for MeCP2, both MeCP2 isoforms were most likely precipitated by ChIP. An unexpected non-specific effect of increasing the homologous pairing was observed following transfection of either MDWT or MDMT decoys using the single copy probes to acrocentric chromosomes (GABRB3 and LSI 22). This result could be because of the subtle effects of the decoys or transfection on the binding of MBD1 or MBD2 to endogenous CpG sites (Fig. 6B), as MBD2 has been shown to repress rRNA transcription and could influence the nucleolar organization (68Go). These effects were neither methylation-specific by ChIP nor 15q11–13-specific by FISH, suggesting that MDMT was essential for controlling the non-specific effects of the transfection and decoy approach. Although our results strongly implicate MeCP2 in the process of homologous pairing of 15q11–13 domains, we cannot exclude the possibility of additional methylation-specific or non-specific effects of the decoy transfection explaining our results. The relatively subtle effect of the MeCP2 decoy on 15q11–13 pairing in SH-SY5Y cells suggests that factors in addition to MeCP2 may be important in the interaction.

Mecp2-deficient mouse models of RTT recapitulate the disorder but with a milder phenotype, as hemizygous male mice are more similar to heterozygous female RTT patients in onset and severity (69Go,70Go). Interestingly but unfortunately, no evidence for homologous pairing of the 15q11–13 syntenic region in mouse (7qB4) was observed in either wild-type or Mecp2-deficient mouse brain at any developmental stage (Supplementary Material, Fig. S4 and Table S2). The most likely explanation for the discrepancy is that mouse 7qB4 is not adjacent to ribosomal DNA (rDNA) genes as it is for acrocentric chromosome 15 in human, as the placement of the 15q11–13 domain close to rDNA genes occurred during primate evolution (71Go). Alternately, the ~1 Mb region upstream of the MeCP2 binding SNRPN promoter containing the recently duplicated maternal imprinting conrol region (72Go) that is not conserved in mouse chromosome 7B4 but is highly conserved in the chimpanzee (UCSC human genome browser, Chimpanzee Genome Sequence Consortium) (73Go) could explain the discrepancy between species. Interestingly, Mecp2 deficiency in mouse results in reduced expression of both UBE3a/Ube3a and GABRB3/Gabrb3, although less significantly than that observed in human RTT or autism brain (17Go). Perhaps MeCP2 can act in both cis as a long-range regulator of chromatin and trans when 15q11-13 homologs are close. This possibility is supported by the formation of oligomeric chromatin suprastructures by MeCP2 in vitro (65Go) but remains to be directly investigated. The observation that MeCP2 decoy specifically blocked the association of GABRB3 but not CEP15 would also support a specific role for MeCP2 in 15q11–13 rather than nucleolar organization.

Although the mechanism of homologous pairing of imprinted 15q11–13 domains has not been fully characterized, at least one part of the pathway must involve an allele-discrimination step. Allele-specific methylation patterns are found throughout 15q11–13 (42Go,74Go,75Go), with the most stable methylated sites at the 5' end of the maternal SNURF/SNRPN within the imprinting control region (76Go). Our ChIP results demonstrate that MeCP2 binds to this methylated CpG island and binding is increased following SH-SY5Y differentiation. The reduced 15q11–13 pairing in brain samples from patients with RTT and autism may therefore be due to the defects in MeCP2 expression in these samples. As MeCP2 has been shown to be involved in silent chromatin looping for both Igf2/H19 (77Go) and Dlx5/Dlx6 (61Go) imprinted domains, perhaps loss of MeCP2 binding to the imprinting control region and other differentially methylated regions of 15q11–13 results in abnormal loop structures and nuclear mislocation of both alleles of UBE3A and GABRB3. We have recently described expression defects of UBE3A and GABRB3 in autism and RTT brain samples, consistent with this model (17Go). Although the possibility remains that the 15q11–13 nuclear organization changes observed here may be unrelated to the expression changes of UBE3A and GABRB3 or to the disease etiology, the model of chromatin looping and transcriptional activity is a testable one. Clearly, much additional work lies ahead in understanding the mechanism of homologous pairing of chromosomes in postnatal brain as well as the downstream effects on gene expression within the 15q11–13 region. Our results suggest, however, that the pathogenic mechanisms of overlapping human autism-spectrum disorders with different underlying genetic causes may intersect at the developmentally controlled organization of oppositely imprinted 15q11–13 domains in the postnatal brain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Tissue culture
SH-SY5Y neuroblastoma cells (ATCC) were grown in complete minimal essential media with 15% fetal calf serum. Cells were seeded onto two-chamber glass slides treated with poly-D-lysine and grown on slides until 30–50% confluent. Cells were fixed either before (untreated) or 72 h after the addition of 16 nM PMA (PMA treated) for 15 min in Histochoice (Ameresco) then washed in 1xPBS/0.5% Tween for 5 min and stored in 70% ethanol at –20°C.

Brain tissue microarray
The paraffin-embedded tissue microarray described previously (16Go) containing triplicate frontal cortex (Brodman area 9, layers III–V) was sectioned at 5 µm onto glass slides. Slides were baked overnight at 55°C, then placed in four 5 min washes with xylene, then two 5 min washes with 100% ethanol and then 1 h at 95°C in antigen retrieval solution (DAKO). Slides were then post-fixed in Histochoice for 90 min and then washed 5 min in 1x PBS. A mouse brain tissue microarray was constructed containing multiple Mecp2+/y, –/y, +/+, –/+ cerebrum samples (developmental ages shown in Supplementary Material, Table S2) obtained by mating Mecp2tm.1.Bird/+ females (Jackson Labs) to C57BL/6 wild-type males as described previously (78Go).

FISH
Slides were dehydrated in 70, 90 and 100% ethanol (10 min each) and then dried at 50°C. A probe mixture containing 1 µl each probe (Vysis, Inc.), 2 µl ddH2O and 7 µl LSI/WCP buffer (Vysis, Inc.) was warmed to 37°C, then added to the slide, coverslipped and sealed with rubber cement. Probe and cells were simultaneously denatured at 80°C for 1.5 min (for SH-SY5Y cells) or 85°C for 2 min (for tissue micoarray slides) on a slide cycler (Hybaid). Slides were incubated overnight at 37°C, then washed in 50% formamide/50% 2xSSC thrice for 5 min, 0.5xSSC for 5 min and 0.5xSSC/0.1% IGEPAL for 5 min, all at 46°C and pH 7.6. To the slides 250 µg/ml RNase was added, coverslipped and incubated at 37°C for 30 min, then 5 min in 1xPBS and air dried. Slides were mounted with 5 µg/ml DAPI in Vectrashield (Vector Laboratories), coverslipped and sealed with nailpolish. Mouse BAC clones for Gabrb3 (RP23-24D4), Snrpn (RP24-275J20) and Ptgs1 (RP23-274M8) were labeled with biotin or digoxigenin by nick translation and detected as described previously (33Go).

Fluorescence microscopy
Slides were analyzed on an Axioplan 2 fluorescence microscope (Carl Zeiss, Inc., NY, USA) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ, USA), appropriate fluorescent filter sets, and automated xyz stage controls. The microscope and peripherals were controlled by a Macintosh running IPLab Spectrum (Scanalytics, Vienna, VA, USA) software with Multiprobe, Zeissmover and 3D extensions. Images were captured for blue, green and red filters at one edge of the specimen, then repeated at 0.4 µm sections through the depth of the tissue. Each image stack was digitally deconvolved to remove out-of-focus light using HazeBuster software (Vaytek, Fairfield, IA, USA). Following haze removal, image stacks for each fluorophore were merged and stacked to create a two-dimensional image representing all of fluorescence within the section. Scoring of FISH signals was perfomed manually and results are averages of scoring performed both blinded (in which the individual scoring did not know the identity of the samples) and unblinded, as no evidence for bias was observed (Supplementary Material, Table S1).

MeCP2 decoy transfections
MeCP2 decoy and control decoy were obtained commercially (GeneDetect.com). Both mutant (5'-TAATCTAGTCTAGACTAGATTA-3') and wild-type (5'-TAATCCGGTCTAGACCGGATTA-3') double-stranded phosphorothioate oligodeoxynucleotides were treated with HpaII methylase overnight to methylate the CpG sites. The methylase-treated decoys, MDWT (MeCP2 decoy wild-type) and MDMT (MeCP2 decoy mutant control), were digested with HpaII and analyzed by PAGE to confirm methylation.

SH-SY5Y cells were grown on two-chamber glass slides treated with poly-D-lysine and transfected with decoy mixture (100 µl per chamber): 92 µl serum free media, 3 µl Fugene 6 (Roche) and 5 µl methylated decoy (containing 1 µM of either MDWT or MDMT) which was incubated at room temperature for 30 min before addition to slides. Twelve hours after transfection, cells were treated with 16 nM PMA and fixed 72 h later, as described previously.

Chromatin immunoprecipitation
Chromatin was prepared from SH-SY5Y cells and purified by urea gradient centrifugation as described previously (79Go,80Go). Immunoprecipitation, reverse crosslinking and PCR amplification were performed as described previously (81Go) with some modifications. For each experiment, 150–200 µg of chromatin was digested into ~5 kb fragments with Sac1 (New England Biolab) and precleared first by incubation with appropriate agarose beads (PrecipHen agarose, Aves labs or protein A/G agarose, Pierce) alone, then with appropriate preimmune serum (preabsorbed IgY, rabbit IgG, mouse IgG) followed by agarose beads. Precleared chromatin was divided (~30 µg per tube) and incubated overnight with 5 µg of either C-terminal anti-MeCP2 (raised in chicken to C-terminal peptide N-RPNREEPVDSRTPVTERVS-C, Aves Labs) or preabsorbed IgY as a control for non-specific binding; C-terminal anti-MeCP2 (rabbit commercial, Upstate); anti-MBD1 (Affinity bioreagents), anti-MBD4 (Imgenex), or rabbit IgG control; anti-MBD2 and anti-MBD3 (Imgenex) or mouse IgG control. Antibody incubations were followed by additional incubation for 4–6 h with 40 µl of agarose beads. Equal amounts of precleared chromatin were processed without IP as total input control. Immunoprecipitates collected by centrifugation were washed, then digested with 50 µg/ml DNase free RNaseA for 30 min at 37°C, followed by SDS/proteinase K digestion and subjected to phenol/chloroform extraction before ethanol precipitation with glycogen. One-twentieth of the DNA from each IP reaction was PCR amplified in reactions containing 2.5 U of TaKaRa LA Taq (TaKaRa), 1xGC buffer I or II, dNTP mix (2.5 mM each) and 0.2 µM primers of either Pr 291 and Pr 292 (5'-actgccatagcctcctcgcctc-3' and 5'-cttgctgttgtgccgttctgcc-3') specific to the SNURF/SNRPN promoter within the 15q11–13 imprinting control region or Pr 279 and Pr 280 (5'-ccaatctcagtcccttccccc-3' and 5'-gtttctctccgcccgtcttc-3') specific to the GAPDH promoter region using one cycle of 95°C for 5 min, 30–35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, with a final cycle of 72°C for 7 min. PCR products were resolved by agarose gel electrophoresis, stained with Sybr Gold (molecular probes) and intensities of the PCR bands were quantified using GelExpert software (Nucleotech).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors thank K. Ehmsen, D. Braunschweig and R. Samaco for technical assistance and M. Lalande for critical reading of the manuscript. This work was supported in part by the U.C. Davis MIND Institute, the Rett Syndrome Research Foundation and the NIH (1R01HD/NS41462). Human tissue samples were generously provided by the Autism Tissue Program, the University of Maryland Brain and Tissue Bank for Developmental Disorders (supported by NIH N01-HD-1-3138), Harvard Brain Tissue Resource Center (supported in part by PHS MH/NS 31862) and M. Lalande.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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