Regulation of alternative splicing by histone modifications

Abstract

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery.

Fig. 1

Splicing-specific histone modifications. (A) Schematic representation of the human FGFR2 gene. Exon IIIb (red) is included in PNT2 epithelial cells, exon IIIc (black) is included in hMSCs. Square dots indicate oligonucleotide pairs used in analysis. (B) Levels of FGFR2 exon inclusion relative to GAPDH in PNT2 (red) or hMSCs (black) determined by quantitative polymerase chain reaction (PCR). (C to H) Mapping of H3-K27me3 (C), H3-K36me3 (D), H3-K4me3 (E), H3-K4me1 (F), H3-K9me1 (G), and H3-K4me2 (H) in FGFR2 in PNT2 (red) and hMSC (black) cells by quantitative chromatin immunoprecipitation. The percentage of input was normalized to unmodified H3. Values represent means ± SEM from four to six independent experiments. *P < 0.05, **P <0.01, Student's t test.

Fig. 2

Modulation of alternative splicing by his-tone modifications. (A, E, and I) H3-K36me3 levels after SET2/SETD2 modulation [(A) and (E)] and H3-K4me3 after ASH2 overexpression (I). Arrows indicate transfected cells. Scale bar, 10 mm. (B to D, F to H, and J to L) Quantitative reverse transcription PCR (RT-PCR) analysis of exon ratios after overexpression of Flag-SET2 [(B) to (D), SET2], down-regulation of SETD2 [(F) to (H), siSETD2], or over-expression of Flag-ASH2 [(J) to (L), ASH2] in PNT2 (red) or hMSC cells (black). Ratios are as follows: exon IIIb/IIIc for FGFR2, exon v6/e2 for CD44, and exon e6/e7 for GAPDH. Values represent means T SEM of percentages relative to EGFP or siCyclophilin B used as controls from four to six independent experiments. **P <0.01, Student's t test compared to control.

Fig. 3

MRG15 modulates alternative splicing. (A and B) Chromatin immunoprecipitation of MRG15 along FGFR2 (A) and CD44 (B) in PNT2 (red) and hMSC (black) cells. Input was normalized to unmodified H3. The RPL13a promoter was a positive control. Values represent means ± SEM from three to six independent experiments. (C to F) Quantitative RT-PCR analysis of exon ratios after overexpression of EGFP-MRG15 [(C) and (D), MRG15] or down-regulation of both MRG15 and MRGX [(E) and (F), siMRG] in PNT2 (red) or hMSC (black) cells. Ratios are as follows: exon IIIb/IIIc for FGFR2 and exon v6/e2 for CD44. Values represent means ± SEM of percentages relative to EGFP or siCyclophilin B used as controls from five independent experiments. *P<0.05, **P<0.01, Student's t test compared to control.

Fig. 4

H3-K36me3 recruits PTB to exon IIIb via MRG15. (A) Immunoblot detection of PTB and MRG15 after immuno-precipitation of PTB. (B) Reciprocal coimmunoprecipitation of over-expressed MRG15-V5/His and PTB-HA in Hela cells. (C to E) RNA immunoprecipitation of PTB, MRG15, or Sm proteins in FGFR2 exons IIIb and IIIc in PNT2 (red) or hMSC (black) cells. Values represent means ± SEM from three independent experiments. (F and G) Overexpression of MRG15 (F) or SET2 (G) in PNT2 (red) or hMSC (black) cells for 48 hours before RNA immunoprecipitation of PTB. Values represent means ± SEM from three independent experiments. (H and I) Quantitative RT-PCR analysis of exon ratios in PNT2 cells depleted of MRG15 and MRGX (siMRG, yellow) or cyclophilin B (siCycB, red) after overexpression of SET2 or EGFP. Ratios are as follows: exon IIIb/IIIc for FGFR2 (H) and exon v6/e2 for CD44 (I). Values represent means T SEM from five independent experiments. *P < 0.05, **P < 0.01, Student's t test compared to control.

Fig. 5

Genome-wide identification of SETD2, MRG15, and PTB alternative splicing events in hMSC cells. (A) Scatterplot of AS events in MRG15- (siMRG) or PTB-depleted cells (siPTB) as compared to small interfering RNA control (siCtrl) transfected cells. The cutoff is ≥15% change in exon usage. Red indicates codependent splicing events. (B) Venn diagram of PTB- and MRG15-dependent events with ≥15% change in exon usage. (C) Percentage of MRG15/PTB codependent events that are also sensitive to SETD2 down-regulation. (D) An adaptor system for reading histone marks by the splicing machinery, consisting of a histone mark signature, a chromatin-binding protein (MRG15), and a splicing regulator (PTB).