15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain

Abstract

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

Figure 1.

Dynamic 15q11.2–13.3 chromatin decondensation during neuronal maturation. (A) 4C analysis of 15q11.2–13.3 chromatin structure in developing neurons shows developmentally regulated chromatin decondensation in maturing neurons. 15q11.2–13.3 loci that contact the PWS-IC (red box) are shown as a series of log 2 signal peaks from representative hybridization of 4C libraries isolated from developing SH-SY5Y neurons (row 3) and untreated SH-SY5Y neuroblasts (row 5). 4C peaks corresponding to sites of PWS-IC interaction were determined using a bioinformatic analysis of three independent replicate experiments for both PMA-treated neurons and untreated neuroblasts and are shown as a series of black bars above the corresponding peaks (rows 2 and 4). MeCP2-binding sites determined from previous studies are shown as a series of purple bars in row 1. 15q11.2–13.3 was subdivided into regions A–C based on Giemsa banding patterns in metaphase chromosomes (top row). Log 2 signal ratios are shown as red and green histograms in the UCSC Genome Browser with known genes and transcripts shown in blue font (row 6). A black line at a log 2 signal ratio of 1 is included to enable comparison of peaks in rows 3 and 5. Segmental duplications are shown as a series of colored bars below chromosome bands. (B). A high-resolution example of PWS-IC interactions flanking the HBII-85 snoRNA cluster in neuroblasts (bottom histogram) that are lost during neuronal differentiation (top histogram). Arrows show PWS-IC-binding interactions. (C). PWS-IC interactions shown as black lines above the log 2 signal peaks with the autism candidate gene GABRB3 are also altered by neuronal differentiation. PWS-IC interactions are indicated by red arrows.

Figure 2.

PWS-IC- and MeCP2-bound sites map to CHRNA7 and CHRFAM7A. 4C analysis reveals PWS-IC interaction sites that are also bound by MeCP2 in 15q13.3. (A) A 250 kb scale view of CHRNA7 chromatin structure reveals two sites (arrows) that are bound both by the PWS-IC (PWS-IC peaks) and MeCP2 (top bars) as determined by previous ChIP-chip analysis. 4C interactions shown as log 2 signal ratio peaks of differentiated SH-SY5Y 4C libraries identify one bivalent site 50 kb upstream of CHRNA7 near the AK097050 transcript in neurons undergoing differentiation (upper histogram) and another site ∼30 kb downstream. (B) Overlap between MeCP2-bound and PWS-IC-bound sites was also observed within an intron of CHRFAM7A (left arrow) and upstream of the translational start site and near the AK127208 transcript (right arrow) in maturing neurons. (C) MeCP2 knockdown by siRNA in SH-SY5Y cells reduces interaction of CHRNA7 with the PWS-IC. Insets show PWS-IC interactions in neurons with normal levels of MeCP2 (top histogram) as peaks compared with neurons with reduced MeCP2 levels (bottom histogram) with diminished PWS-IC interaction signals.

Figure 3.

PWS-IC interaction with CHRNA7 is validated by FISH. Two-color FISH analysis confirms the interaction of the PWS-IC with CHRNA7. (A) BAC clones complementary to the PWS-IC (RP11-125E1) and CHRNA7 loci (RP11-717124 as shown on the 15q11.2–13.3 map were differentially detected as green and red fluorescent signals, respectively). (B) PWS-IC and CHRNA7 signals were considered associated if there was a spatial overlap or contact between the red and green signals. One hundred nuclei from undifferentiated and maturing (SH − SY5Y + PMA) neurons were scored for signal overlap in each allele and the possible combinations (middle diagram) and were graphed as a percentage of total nuclei (left bar graph). Representative images of each PWS-IC/CHRNA7 allelic combination are shown in the right panel with scale bars.

Figure 4.

CHRNA7 transcript levels decline significantly with age in the human cortex. QRT–PCR analyses of CHRNA7 expression in normally developing control (CTRL, N = 20), Rett (RTT, N = 7) and autism (AUT, N = 12) were performed on RNAs isolated from frozen, post-mortem Brodmann area 9 cortices using CHRNA7- and GAPDH-specific primers. Fold change of CHRNA7 expression relative to GAPDH was calculated using the comparative Ct method for each sample. CHRNA7 expression declines rapidly from 4.0-fold relative to GAPDH in 4-month-old cortex to ∼0.4-fold in 57-year-old cortex. Analysis of CHRNA7 expression levels was done using a one-way ANCOVA with age as a covariate showing that age has a significant relationship to CHRNA7 expression (P< 0.0001).

Figure 5.

CHRNA7 transcripts are significantly reduced in Rett and autism corticies. QRT–PCR analyses identify significant reductions in CHRNA7 expression in Rett and autism frontal cortices. RNAs were isolated from Brodmann area 9 of patients with classic Rett syndrome (RTT, N = 7) and autism patients (AUT, N = 11). RNAs were amplified and quantified using CHRNA7- and GAPDH-specific primers, and CHRNA7/GAPDH fold changes were calculated using the comparative Ct method and graphed, and normalized to the typically developing controls (CTRL, N = 20). Significant reductions (P< 0.05) in CHRNA7 transcripts are observed in both RTT and AUT samples compared with CTRL while controlling for the effect of age. Fold changes for RTT and AUT samples are normalized to CRTL samples and error bars correspond to standard error of the mean. P-values are derived from ANCOVA with age as a covariate.