Global regulation of alternative splicing during myogenic differentiation

Abstract

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.

Figure 1.

Characterization of validated splicing transitions associated with C2C12 myoblast differentiation. (A) Phase-contrast micrographs showing a time course of C2C12 differentiation. (B) The number of splicing events (out of 117 total validated events) that undergo splicing transitions of ≥20 percentage points. (C) Summary of the different types of validated splicing transitions included within the data set of 117 splicing transitions. (D) GO analysis for significantly (P ≤ 0.05) enriched molecular functions in validated splicing transitions of ≥20 percentage points.

Figure 2.

Most AS events common to mouse and quail myoblasts undergo conserved transitions during differentiation. (A) Representative RT–PCR results for seven splicing transitions conserved between C2C12 (mouse) and QM7 (quail) myoblast differentiation. Undifferentiated (U) and differentiated (D) cultures are indicated. The percentage alternative exon inclusion (% inclusion) is indicated in undifferentiated and differentiated C2C12 and QM7 cells. (B) Summary of splicing transitions conservation between C2C12 and QM7 cells. See Supplementary Table S2 for a full list of all events tested between C2C12 and QM7 cells.

Figure 3.

Most splicing transitions are dependent upon myogenic differentiation. (A) Phase-contrast micrograph of vehicle (DMSO) and BDM (15 μM) treated C2C12 cells 120 h following induction of differentiation. (B) Western blots of whole cell lysate from DMSO or BDM treated C2C12 cells. Myogenin staining was used to measure myogenic differentiation progression and p27 staining was used to assess cell-cycle exit. ‘Asterisk’ indicates ∼60 kDa non-specific band detected by p27 antibody. (C) Four groups each depicting four representative examples of splicing transitions distinguished based on myogenic differentiation dependence. BDM (dashed lines) or DMSO vehicle (solid lines) was added to media at Hour 0. Group i exhibited a complete block of splicing transitions following addition of BDM. Group ii exhibited partial reversion of splicing transitions to undifferentiated (−24 h) state upon treatment with BDM. Group iii continued to undergo splicing transitions after BDM treatment, but at a reduced level compared to DMSO. Group iv events were not affected by addition of BDM. Splicing events depicted (in the order: black, green, red, orange) are as follows: Group i: Dtna_78,93; Atp2b1_87; Lrrfip2_93,102; Anxa7_66. Group ii: Dguok_100; 4632411B12R Rik_78; Mpp6_42; Azi2_47. Group iii: Capzb_113; Art3_30; Akap13_54; Bin1_45. Group iv: R3hdm_42; 5830434P21Rik_82; 5230400G24Rik_60; Pkp4_129.

Figure 4.

Motif enrichment and conservation in intronic regions flanking regulated variable regions. Pentameric motifs that are significantly enriched (upper panel) within the flanking intronic sequences of exons exhibiting AS changes during C2C12 differentiation or conserved (lower panel) among seven other mammalian species. Motifs were identified using a first-order Markov background model (corrected P < 0.05, motifs in italics indicate corrected P < 0.0001). Intronic regions analyzed included the first and last 250 nt of the upstream and downstream introns, excluding the first 9 nt and last 30 nt of the introns. Motifs matching the recognition sequences of known splicing regulators are indicted. Only motifs with significance P < 0.05 (standard text) or P < 0.0001 (italic text) are shown in the figure. For a complete list of all enriched and conserved motifs, see Supplementary Tables S3 and S4, respectively.

Figure 5.

Regression analysis identified enriched motifs associated with temporal splicing transitions. Regression analysis was used to identify correlations between enriched motifs and specific temporal patterns of splicing transitions. The enrichment of each motif within the various intronic regions was regressed against the magnitude of each splicing transition (% of total change) at −24, 0, 12, 24, 48, 72 and 120 h relative to differentiation induction. Green denotes positive correlations (motif associated with increased inclusion of alternative region during differentiation); red denotes inverse correlations (decreased inclusion of alternative region during differentiation). Early (before 24 h), late (after 24 h) and continuous refers to period in the differentiation time course to which a given motif is most strongly correlated. For a full display of all regression analysis heatmap data see Supplementary Figure S1.

Figure 6.

Clusters of splicing transitions with similar temporal patterns exhibit enrichment of specific motifs. Splicing transitions that share similar temporal patterns were grouped together in clusters using affinity propagation analysis. Each cluster contains a single ‘exemplar’ event (red), which typifies the temporal behavior of that cluster. The remaining events in each cluster are plotted in various shades of gray, where darker gray represents a larger maximum difference in inclusion level across all time points. (A) Four major clusters (n ≥ 10) representing 102 out of 111 events analyzed by affinity propagation analysis. A total of six clusters were identified in all; the two remaining contain ≤8 events and are shown in Supplementary Figure S2. (B) Motif enrichment and conservation analysis identical to that described in Figure 4A was performed on the four clusters shown in Figure 5A. Significantly enriched (corrected P < 0.05, italics indicate corrected P < 0.0001) motifs are shown. Motifs matching the recognition sequences of known splicing regulators are indicted. For a list of all cluster-specific enriched motifs, see Supplementary Table S5. For a full list of all cluster specific enrichment and conservation date see Supplementary Tables S6 and S7, respectively.

Figure 7.

Western blot analysis shows dynamic regulation of candidate splicing factors during C2C12 differentiation. Steady-state nuclear and cytoplasmic protein levels of MBNL, CELF, FOX, PTB, hnRNP C and hnRNP L splicing regulators during C2C12 differentiation. TBP and GAPDH serve as nuclear and cytoplasmic markers, respectively. Ponceau S staining serves as a loading control.