Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing

Abstract

In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, independently of the calcium/calmodulin protein kinase IV pathway. We show that this exon responds to RNA polymerase II elongation, because its inclusion is increased by a slow polymerase II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyper-acetylation restricted to an internal region of the NCAM gene surrounding the alternative exon. This intragenic histone hyper-acetylation is not paralleled by acetylation at the promoter, is associated with chromatin relaxation, and is linked to H3K36 tri-methylation. The effects on acetylation and splicing fully revert when the depolarizing conditions are withdrawn and can be both duplicated and potentiated by the histone deacetylase inhibitor trichostatin A. Our results are consistent with a mechanism involving the kinetic coupling of splicing and transcription in response to depolarization through intragenic epigenetic changes on a gene that is relevant for the differentiation and function of neuronal cells.

Fig. 1.

Depolarization promotes NCAM E18 skipping in new transcripts independently of protein synthesis and of CaMKIV inhibition. N2a cells were treated with different concentrations (A) or 60 mM KCl (B, C, and E–H) for 16 h. (D) Rat cultured hippocampal neurons were treated with 40 mM KCl for 20 h or left untreated. After these treatments, cells were harvested, total RNA prepared, and alternative splicing for the indicated genes was assessed with specific primers by a 30-cycle radioactive RT-sqPCR (A–C and G) or real-time RT-qPCR (D–F and H) as described in Materials and Methods. In A, half the cells were harvested and the other half were incubated in normal medium for another 20 h (Recovery). Treatments in E–H were performed in the presence or absence of the transcription inhibitor actinomycin D (Act D; 5 μg/mL), the protein inhibitor CHX (5 μg/mL), or the CaMKIV inhibitor KN93 (20 μM) as indicated. (CPEB4, cytoplasmic polyadenylation element binding protein 4; NR1, NMDA receptor 1; Ctl., control; Dep., depolarization.) Bars show means ± SD corresponding to 2 or 3 independent experiments.

Fig. 2.

Transcription by C4 “slow” RNA pol II increases E18 inclusion. An NCAM splicing reporter mini-gene was co-transfected in N2a cells with an empty plasmid (-) or plasmids expressing WT or C4 α-amanitin-resistant RNA pol II large subunits. Cells were treated with α-amanitin to inhibit endogenous polymerase. Splicing patterns were assessed by a 30-cycle radioactive RT-sqPCR. Left and right panels correspond to independent experiments. Bars show means ± SD (n = 2).

Fig. 3.

Depolarization triggers reversible general histone acetylation. N2a cells were treated for 6 to 8 h with 60 mM KCl (Depol) or left untreated (Control), and Western blot with antibodies for acetyl-histones H3 and H4, and total histone H3, were performed as indicated in Materials and Methods. Quantification of the increase in histone acetylation corresponding to the pooled data of 2 independent experiments is shown (means ± SD; n = 2). After the treatment, some cells were incubated for 20 more hours in normal medium (Recovery).

Fig. 4.

Depolarization triggers local changes in histone modifications and chromatin structure assessed by nChIP. (A) Scheme of the ncam gene showing the distribution of qPCR amplicons. (B) Levels of H3K9 acetylation at the regions indicated in A, corresponding to untreated N2a cells. (C) Increase of H3K9 acetylation in the same regions in response to depolarization with 60 mM KCl for 4 to 6 h. Inset, qPCR amplification curves from control (C) and depolarized (D) samples corresponding to a non-responding region (i.e., promoter) and a region that shows increased acetylation levels after depolarization (i.e., intron 17) (D) Negative control showing no changes in H3K9 acetylation on the FN gene at the EDI alternative exon. Means and SD correspond to 2 or 3 independent experiments. (E) Representative levels of H3K36 tri-methylation at the regions indicated in A, corresponding to untreated N2a cells. (F) Increase of H3K36 tri-methylation in the same regions in response to depolarization with 60 mM KCl for 6 h. Means and SD correspond to 2 independent immunoprecipitations of 1 experiment. (G) Scheme of the MspI accessibility assay used (Upper) and bar graph depicting the changes in chromatin accessibility (Lower). Cells were treated for 5 h with 60 mM KCl (Dep) or 1 μM TSA or left untreated (Ctl). The accessibility index was evaluated (see Materials and Methods) for the NCAM exon 18 region and a promoter region. Results are expressed relative to value obtained in each amplicon for the control cells.

Fig. 5.

Depolarization and histone acetylation increase RNA pol II processivity. (A) Scheme showing the proximal and distal amplicons used for qPCR assessment. (B) Increase in D/P pre-mRNA accumulation. Cells were treated for 4 to 6 h with high KCl (Depol) or TSA (3.3 μM). Bars correspond to the normalized means and SEs for the pooled data of 3 (Control and Depol) or 2 different experiments (TSA). The table below shows the results of a 2-way ANOVA used to analyze statistical significance, considering the different treatments as one factor and the different experiments as the other factor (no significant effect of experiment or interaction between the two was found). (C) Depolarization after recovery in normal medium. Cells were treated for 4 h and recovered in normal medium for 20 more hours. Bars correspond to the normalized means and SEs of one experiment in triplicate.

Fig. 6.

TSA treatment mimics and potentiates the depolarization effect on NCAM E18 alternative splicing. (A) N2a cells were treated for 16 h with 60 mM KCl (Depolarization), 3.3 μM TSA, or both, or left untreated (n = 2 for control and n = 3 for depolarization treatment). (B) N2a cells were treated with 3.3 μM TSA or vehicle in the presence of CHX (n = 2). (C) N2a cells were treated for 6 h with 60 mM KCl, 10 nM TSA, or both, or left untreated (n = 3; **Student t test, P < 0.0001 vs. control, P < 0.005 vs. TSA, P < 0.05 vs. depolarization). NCAM E18 splicing patterns were assessed as before (means ± SD).