Neuron-specific impairment of inter-chromosomal pairing and transcription in a novel model of human 15q-duplication syndrome

Abstract

Although the etiology of autism remains largely unknown, cytogenetic and genetic studies have implicated maternal copy number gains of 15q11-q13 in 1-3% of autism cases. In order to understand how maternal 15q duplication leads to dysregulation of gene expression and altered chromatin interactions, we used microcell-mediated chromosome transfer to generate a novel maternal 15q duplication model in a human neuronal cell line. Our 15q duplication neuronal model revealed that by quantitative RT-PCR, transcript levels of NDN, SNRPN, GABRB3 and CHRNA7 were reduced compared with expected levels despite having no detectable alteration in promoter DNA methylation. Since 15q11-q13 alleles have been previously shown to exhibit homologous pairing in mature human neurons, we assessed homologous pairing of 15q11-q13 by fluorescence in situ hybridization. Homologous pairing of 15q11-q13 was significantly disrupted by 15q duplication. To further understand the extent and mechanism of 15q11-q13 homologous pairing, we mapped the minimal region of homologous pairing to a ∼500 kb region at the 3' end of GABRB3 which contains multiple binding sites for chromatin regulators MeCP2 and CTCF. Both active transcription and the chromatin factors MeCP2 and CTCF are required for the homologous pairing of 15q11-q13 during neuronal maturational differentiation. These data support a model where 15q11-q13 genes are regulated epigenetically at the level of both inter- and intra-chromosomal associations and that chromosome imbalance disrupts the epigenetic regulation of genes in 15q11-q13.

Figure 1.

Characterization of SH-SY5Y neuronal cells containing an extra maternal human chromosome 15. (A) A schematic diagram showing the construction of SH-SY5Y microcell hybrids [SH(15M) cell] containing an extra maternal copy of human chromosome 15. Transfer of a maternal human chromosome 15 from mouse A9 cells to SH-SY5Y cell was performed by MMCT technology. (B) RFLP analysis to confirm presence of donor chromosome 15 in SH(15M) cells is shown. DNA from introduced maternal human chromosome 15 donor A9 cells (lane 1), SH-SY5Y cells (lane 2), SH(15M)-1, 2, 3 cells (lanes 3–5) and mouse A9 cells (lane 6) was amplified by PCR using primers that span a PvuII polymorphism. Both chromosome 15 copies in the host SH-SY5Y cells were digested by PvuII, but the donor chromosome 15 was undigested. SH(15M) cells contained both digested and undigested fragments. (C) Results from DNA-FISH analysis of SH(15M) cells. Metaphase chromosomes from SH(15M) were hybridized in situ with Vysis LSI GABRB3 (red) and CEP15 (green) probes. The arrowheads indicate three copies of chromosome 15.

Figure 2.

Analysis of 15q11–q13 transcript levels in experimental model of dup15q syndrome. (A) Physical map of the imprinted gene cluster in human chromosome 15q11–q13. PWS-IC is the PWS imprinting center, which is essential for establishment of the paternal epigenetic state of the region. Genes or transcripts (filled boxes) are drawn approximately to scale. Transcriptional direction is indicated by arrowheads and arrows. P, paternally expressed genes; M, maternally expressed genes; B, biallelically expressed genes. (B–I) Summary of quantitative RT–PCR measurements of eight transcripts in 15q11–q13, normalized to the housekeeping genes GAPDH and ACTB. Error bars represent ±SEM. All qRT–PCR analyses were performed on cDNA from PMA-treated differentiated SH-SY5Y cells (WT) and PMA-treated differentiated SH(15M) cells (–3). (B)–(E) are non-imprinted biallelically expressed genes, (F) and (G) are paternally expressed imprinted genes and (H) and (I) are maternally expressed imprinted genes.

Figure 3.

Reduced homologous pairing of GABRB3 alleles as a result of duplicated maternal chromosome 15 in SH-SY5Y cells. (A) Representative image of chr.15–chr.15 localization in SH-SY5Y cells (left panel) and SH(15M) cells (right panel) using Vysis LSI GABRB3 SpectrumOrange/CEP15 SpectrumGreen. The iVision software was used to measure distances between two signals. In case of SH(15M) cells, we measured the shortest inter-chromosomal distances between the three signals. Scale bars: 5 µm. (B) Differences in homologous pairing in SH-SY5Y cells (gray bars) and SH(15M) cells (white bars), as determined by hybridization with Vysis LSI GABRB3 (left panel) and CEP15 (right panel) FISH probes. Pairing was scored as homologous FISH signals with distances ≤2 μm apart. The bars indicate the mean ± SEM of three replicate experiments, in each of which 100–150 nuclei were scored. Significantly fewer SH(15M) cell nuclei showed pairing when the GABRB3 probe was used. In contrast, no significant difference was observed in SH(15M) cells in pairing between alleles detected by the pericentromeric CEP15 probe.

Figure 4.

Increased homologous pairing of GABRB3 alleles during neuronal differentiation is associated with active transcription. (A) Physical map of the imprinted gene cluster in human chromosome 15q11–q13. PWS-IC is the PWS imprinting center, which is essential for establishment of the paternal epigenetic state of the region. Genes or transcripts (filled boxes) are drawn approximately to scale. Transcriptional direction is indicated by arrowheads and arrows. The horizontal bars indicate BAC probes used in the pairing analyses. GABRB3(1) probe indicates the Vysis LSI GABRB3 Spectrum Orange probe. (B) Frequency distribution of distances between homologous alleles at multiple sites along 15q11–q13 for undifferentiated SH-SY5Y cell nuclei, as measured on each BAC probe. Mean distance, open triangle. (C) Frequency distribution of distances between homologous alleles at multiple sites along 15q11–q13 for PMA-treated differentiated SH-SY5Y cell nuclei. Mean distance, open triangle. A high proportion of PMA-treated differentiated SH-SY5Y cells displayed close homologous distances (≤2 µm) at GABRB3 upon neuronal differentiation, as shown by a leftward shift in the distribution. (D) Cumulative distribution of distances between homologous alleles at 0–2 µm. The solid line indicates the undifferentiated SH-SY5Y cells. The dotted line indicates the PMA-treated differentiated SH-SY5Y cells. The closed and open triangles indicate cumulative frequency at 2 µm, respectively. The statistical relevance was assessed by a comparison of the entire histogram of measurement distributions from (B) and (C) using two non-parametric tests, namely Mann–Whitney's U-test and Kolmogorov–Smirnov test. P-values from Mann–Whitney's U-test are as indicated. Sample sizes for each experiment ranged from 100 to 210. (E) Differences in homologous pairing in the undifferentiated SH-SY5Y cells (gray bars) and the PMA-treated differentiated SH-SY5Y cells (white bars), as determined by hybridization with Vysis LSI GABRB3 FISH probes. Pairing was scored as homologous FISH signals with distances ≤2 μm apart. The bars indicate the mean ± SEM of three replicate experiments, in each of which 100–150 nuclei were scored. (F) The PMA-treated differentiated SH-SY5Y cells treated for 4 h with the transcriptional inhibitor, α-amanitin, were hybridized with Vysis LSI GABRB3 (left panel) and CEP15 (right panel) FISH probes and signals measured. Transcriptional inhibition resulted in a significant reduction in the homologous pairing at GABRB3, but not at CEP15. Student's t-test.

Figure 5.

Homologous pairing of 15q11–q13 maps to the 3′ region of GABRB3. (A) Physical map of the cluster of GABA_AR subunit genes in 15q11–q13. The genes are drawn approximately to scale. The direction of transcription is indicated by arrows. The BAC probes used in the pairing analyses are shown by horizontal bars. (B) Frequency distributions of distances between homologous alleles at multiple sites along 15q11–q13 for undifferentiated SH-SY5Y cell nuclei, as measured on each BAC probe. Mean distance, open triangle. (C) Frequency distributions of distances between homologous alleles at multiple sites along 15q11–q13 for PMA-treated differentiated SH-SY5Y cell nuclei. A high proportion of PMA-treated differentiated SH-SY5Y cells displayed close homologous distances (≤2 μm) at GABRB3 upon neuronal differentiation, as shown by a leftward shift in the distribution. Mean distance, open triangle. (D) Cumulative distance distribution for homologous alleles at 0–2 µm. The solid line indicates the undifferentiated SH-SY5Y cells. The dotted line indicates the PMA-treated differentiated SH-SY5Y cells. The closed and open triangles indicate cumulative frequency at 2 µm, respectively. The statistical relevance was assessed by a comparison of the entire histogram of measurement distributions from (B) and (C) using two non-parametric tests, namely Mann–Whitney's U-test and Kolmogorov–Smirnov test. P-values from Mann–Whitney's U-test are as indicated. Sample sizes for each experiment ranged from 100 to 210. One probe from the 3′ region of GABRB3 showed significant differences in homologous intrachromosomal distance frequencies between undifferentiated SH-SY5Y cells (solid line) and the PMA-treated differentiated SH-SY5Y cells (dotted line). In contrast, one probe from the GABRB3 gene body and two probes overlapping GABRA5 and GABRG3 did not show significant homologous pairing. The more centromeric GABRB3(1) probe showed a trend towards homologous pairing that was not significant.

Figure 6.

Disruption of GABRB3 homologous pairing via MeCP2 and CTCF knockdown. (A and E) Results from western blot analysis confirming knockdown of MeCP2 (A) and CTCF (E) proteins in SH-SY5Y cells. (B and F) Results from western blot analysis of cell lysates after siRNA-mediated gene silencing. Both MeCP2 (B) and CTCF (F) proteins experienced ∼80% knockdown. (C and G) Expression of MeCP2 (C) and CTCF (G) proteins in siRNA-treated differentiated SH-SY5Y cells. Scale bars: 10 µm. (D and H) Chr.15–Chr.15 distribution profiles and cumulative frequency curves of MeCP2 (D) and CTCF (H) in siRNA-treated differentiated SH-SY5Y cell nuclei. Mann–Whitney's U-test and Kolmogorov–Smirnov tests were used to determine whether the differences between the curves of the siRNA-treated cells (dotted lines) and the non-targeting siRNA-treated SH-SY5Y cells (solid lines) were significant. The statistical relevance was assessed by comparing whole histograms. P-values from Mann–Whitney's U-test are as indicated. Sample sizes for each experiment ranged from 103 to 105.