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. 2013 Aug 14;32(16):2264-74.
doi: 10.1038/emboj.2013.167. Epub 2013 Jul 26.

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

Ignacio E Schor  1 , Ana Fiszbein, Ezequiel Petrillo, Alberto R Kornblihtt
Affiliations

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation

Ignacio E Schor et al. EMBO J. .

Abstract

Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Differential response of NCAM exon 18 alternative splicing to chromatin-relaxing agents in undifferentiated and differentiated N2a cells. (A) Increase in NCAM E18 inclusion after differentiation of N2a cells assessed by radioactive semi-quantitative RT–PCR or real-time quantitative RT–PCR. Responsiveness of N2a cells (B) and P19 cells (C) to trichostatin A (TSA) in different differentiation stages. For P19 cells, after formation of EBs, the cells are resuspended, and plated in neuronal differentiation medium for the indicated times. Cells were treated with 5 ng/μl TSA for 16 h. (D) Responsiveness of N2a cells to 5-azacytidine (5aC). The indicated cells were treated with 5 μM 5aC for three days. All values are expressed as mean+s.d., relativized to values of control undifferentiated cells. P values correspond to two-tailed Student’s t test (n=3 in all cases).
Figure 2
Figure 2
Differentiated N2a cells show increased repressive histone marks along the NCAM gene. (A) Results of DNA methylation analysis. Following sodium bisulphite modification, genomic DNA was PCR-amplified and 10 clones per sample were sequenced (five are shown). Black and white circles indicate methylated and unmethylated CpG sites, respectively. (B) A scheme of the NCAM gene indicating the amplicons used for nChIP (black thick lines A–J) and for elongation analysis of Figure 3 and Figure 5D (boxes α–η). (C) H3K9me2 analysis along the NCAM gene and the intergenic zone placed 10.6 kbp upstream of the promoter, using native chromatin immunoprecipitation (nChIP). As a control, the housekeeping gene HPRT is shown. Values of two independent immunoprecipitations, relativized to the mean value for HPRT exon 2, are shown for each region. (D) nChIP against H3K9me2 for undifferentiated, differentiated and 5aC-treated differentiated cells. 5aC treatment was as in Figure 1. Values of two independent immunoprecipitations, relativized to the mean value for HPRT exon 2, are shown for each region for Diff. and Diff.+5aC experiments. All values are relativized to those of HPRT exon 4. (E) Quantification of total NCAM mRNA levels. Values (means±s.d.) are expressed in relation to the housekeeping gene HSPCB, and relativized to the mean for undifferentiated cells. P value corresponds to two-tailed Student’s t test (n=3).
Figure 3
Figure 3
Differentiated N2a cells show slower pol II elongation than undifferentiatied cells. Analysis of transcriptional elongation in the indicated regions α (A) and β (B) of the NCAM gene using the method described in Singh and Padgett (2009)). After DRB was washed out to re-initiate transcription, samples were collected at the indicated times for quantification of the pre-mRNA levels using the amplicons depicted in Figure 2B. The values are relativized to the pre-mRNA levels of untreated cells for each experiment. Individual values are shown for each time point. The curves correspond to the mean values at each time point. (C) Analysis of the pre-mRNA level of the regions of NCAM gene depicted in Figure 2B and exon 6 of HPRT is presented as the difference between values at time points of 30 and 0 min after DRB wash-out. Values of individual independent determinations for each time point are shown.
Figure 4
Figure 4
H3K9 methylation and HP1α recruitment contribute to the upregulation of E18 inclusion in differentiated cells. Responsiveness of N2a cells (A) and P19 cells (B) to treatment with BIX before and after differentiation assessed by real-time quantitative RT–PCR. Cells were treated with 1 μM BIX for 3 days. Each individual value corresponds to the mean±s.d. of three replicates. (C) Responsiveness of differentiated N2a cells to treatment with a siRNA against HP1α (siHP1α) or a siRNA control against luciferase (siControl). All values are expressed as mean±s.d., relativized to values of cells transfected with the siControl. The inset shows the steady-state mRNA levels of HP1α measured by real-time RT–PCR, showing a near 50% depletion efficiency. P values correspond to two-tailed Student’s t test (n=3).
Figure 5
Figure 5
Effects of intronic siRNAs on NCAM alternative splicing. (A) Schematic explanation of the TGS-AS mechanism. (B) A scheme of the part of the NCAM gene around exon 18 and the target sites for the intronic double-stranded siRNAs (siI17as and siI18as). (C) Lanes 1 and 2: levels of NCAM E18 inclusion in the presence or absence of siI18as transfection of undifferentiated N2a cells (in the absence of siI18as, a control siRNA against luciferase was transfected). Lanes 3−8: same as lines 1−2, but with the co-transfection of a siRNA against AGO1 (siAGO1), a siRNA against HP1α (si HP1α) or in the presence of BIX. Lane 9: levels of NCAM E18 inclusion in the presence of siI17as transfection of undifferentiated N2a cells. Lanes 10 and 11: same as 1-2 but in differentiated cells. All values are expressed as mean (±s.d.), relativized to values of cells transfected with the siRNA against luciferase. P value corresponds to two-tailed Student’s t test (n=3). (D) H3K9me2 analysis along the NCAM gene in the absence or presence of siI18as transfection using nChIP. The amplicons used are indicated in Figure 2B. Values of two independent immunoprecipitations, relativized to the mean value for HPRT exon 2, are shown for each region. (E) Analysis of transcriptional elongation in the regions α (A) and β (B) of the NCAM gene in the presence or absence of siI18as transfection of undifferentiated N2a cells (in the absence of siI18as, a control siRNA against luciferase was transfected) using the method described in Singh and Padgett (2009). After DRB was washed out to re-initiate transcription, samples were collected at the indicated times for quantification of the pre-mRNA levels using the amplicons depicted in Figure 2B. Values of individual independent determinations for each time point are shown. The inset shows the analysis of pre-mRNA level of the regions of NCAM gene depicted in Figure 2B, presented as Δ(30′−0′) after DRB wash-out relativized to values of cells transfected with siLUC. All values are means+s.d. of duplicates from a representative experiment.
Figure 6
Figure 6
Alternative chromatin-alternative splicing model.
See this image and copyright information in PMC

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