Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells

Abstract

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)₂D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)₂D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)₂D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)₂D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)₂D-mediated induction of transcription.

Figure 1.

Effect of 1,25(OH)₂D on 24-hydroxylase and hnRNPC1/C2 in MG63 cells. (A) (Left) 24-hydroxylase enzyme activity (conversion of ³H-25OHD to ³H-24,25(OH)₂D) in MG63 cells 24 h after addition of vehicle or 1,25(OH)₂D (10 nM). Data are mean ± SD fmol ³H-24,25(OH)₂D_/hr/μg cell protein (n = 3, **P < 0.01, Student's t-test). (Right) Representative high performance liquid chromatography analyses for vehicle (top) and 1,25(OH)₂D (bottom) treatment. (B) qRT-PCR analysis of hnRNPC1/C2 mRNA expression after treatment with vehicle or 1,25(OH)₂D (10 nM) for 12 h. Data are mean ± SD, n = 3. n.s., non-significant. (C) Western blot analysis of 24-hydroxylase and hnRNPC1/C2 with vehicle or 1,25(OH)₂D (10nM) 12 h; N.T., No Treatment. (D) Expression of mRNA for full length CYP24A1, E. CYP24A1-variant 2 (exon 10 skipped), F. CYP24A1-SV (exon 1 and 2 skipped) following treatment with 1,25(OH)₂D (10 nM) up to 12 h. Data are mean ± SD (n = 3, P < 0.05, 1-way ANOVA). Lower panels D and F show schematic illustrations of the full-length, wild-type CYP24A1 and the exon organization of the splice variants described above.

Figure 2.

CYP24A1 DNA and RNA binding of hnRNPC1/C2. (A) Chromatin immunoprecipitation (ChIP) using antibodies to hnRNPC1/C2 (C1/C2) or vitamin D receptor (VDR). Precipitated DNA was PCR amplified with primers to a VDRE-containing region of CYP24A1 promoter and a VDRE-negative region of calponin 1 (CNN1) (negative control). Data are % of input DNA. (B–D) RNA immunoprecipitation (RIP) analyses using antibodies to hnRNPC1/C2 (IgG as a control). Precipitated RNA was PCR amplified with primers encompassing CYP24A1 pre-mRNA, mature CYP24A1 RNA or both as indicated. Data are % of input RNA. Cells were treated with vehicle or 1,25(OH)₂D (10 nM) for 3 h. Data are mean ± SD (n = 3, *P < 0.05, ***P < 0.001, Student's t-test, #, undetectable). n.s. = non-significant.

Figure 3.

Effect of HnRNPC1/C2 knockdown on CYP24A1 transcription and pre-mRNA splicing. MG63 cells were transfected with two hnRNPC1/C2 siRNAs (KD1 and KD2) or scrambled control siRNA (Ctrl). (A) Western blot analysis of CYP24A1 24-hydroxylase and hnRNPC1/C2. Cells were treated with vehicle or 1,25(OH)₂D (10 nM) for 12 h. (B–D) qRT-PCR analysis of mRNA for: (B) wildtype CYP24A1; (C) CYP24A1-variant 2; (D) CYP24A1-SV in KD1, KD2 and Ctrl cells. (E) CYP24A1 exon 10 inclusion by fluorescently labeled RT-PCR. (Left) Inclusion (gray) and exclusion (white) of exon 10. (Right) Gel visualization of PCR products with exon 10 inclusion or skipping. For D–G, cells were treated with vehicle or 1,25(OH)₂D (10 nM) for 12 h. Data are mean ± SD (n = 3, *P < 0.05, **P < 0.01, Student's t-test).

Figure 4.

Alternative splicing of CYP24A1 is modulated by VDRE. Splicing of minigene constructs transfected into MG63 cells containing DNA for exons 9–11 of CYP24A1 linked to promoter DNA from: CMV; CYP24A1 containing two wild-type VDREs; CYP24A1 bearing mutated, non-VDR binding VDREs; VDRE-negative IL-6, ACTB and GAPDH promoters; and VDRE-positive BGLAP. (A) Example of a gel showing bands for correctly spliced minigene transcripts containing exons 9, 10 and 11 (upper band) and alternatively spliced transcripts containing only exons 9 and 11 (skipped exon 10, lower band). (B) Percent exon 10 skipping for each minigene-promoter construct (mean ± S.D., n = 3 separate transfections). * = statistically different from CYP24A1, P < 0.05. Constructs with functional VDRE are highlighted as boxes.

Figure 5.

Differential gene expression by 1,25(OH)₂D or hnRNPC1/2 knockdown. Weighted Venn diagrams depicting overlap of differentially expressed genes: (A) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD; maize), control versus 1,25 (10 nM, 6 h) (Ctrl versus 1,25; grey) or hnRNPC1/C2 knockdown vs 1,25(OH)₂D combined with hnRNPC1/C2 knockdown (KD versus 1,25+KD; blue). (B) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD, maize) or combined 1,25(OH)₂D and hnRNPC1/C2 knockdown (Ctrl versus 1,25+KD, green). (C) Up- and down-regulated differentially expressed genes: (left) Control versus 1,25(OH)₂D (Ctrl versus 1,25) or hnRNPC1/C2 knockdown versus 1,25(OH)₂D combined hnRNPC1/C2 knockdown (KD versus 1,25+KD); (right) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD). (D) Weighted Venn diagrams depicting overlap of differentially expressed genes between Ctrl versus 1,25(OH)₂D and KD versus 1,25(OH)₂D and hnRNPC1/C2 knockdown. Numbers of differentially expressed genes are shown within each segment. Heatmaps show patterns of down-regulated (blue) differentially expressed genes (upper heatmaps) and up-regulated (red) differentially expressed genes (lower heatmaps) for each treatment/knockdown comparison group and the overlapping differentially expressed genes common to those comparisons. * includes CYP24A1.

Figure 6.

Effect of 1,25(OH)₂D and hnRNPC1/C2 knockdown on alternative splicing. (A) Five major types of alternative splicing patterns examined: SE, Skipped Exon; MXE, Mutually Exclusive Exon; A5SS, Alternative 5′ Splice Site; A3SS, Alternative 3′ Splice Site; RI, Retained Intron. (B) Summary of the different differentially expressed genes for each type of alternative splicing event identified by RNA-seq data in MG63 cells with various combinations of no treatment (Ctrl), 1,25(OH)₂D (10 nM 1,25, 6 h) exposure and/or hnRNPC1/C2 knockdown (KD). Run from MATS3.0.8 with significant events at FDR < 0.05, |ΔPSI| ≥ 0.05. * includes CYP24A1.

Figure 7.

Overlap of differentially expressed genes and differentially spliced genes. Venn diagrams depicting overlap of DEG and DSG induced by: (A) hnRNPC1/C2 knockdown (Ctrl versus KD); (B) 1,25(OH)₂D exposure (Ctrl versus 1,25; (10 nM 1,25, 6 h); (C) hnRNPC1/C2 knockdown alone (KD) compared to 1,25(OH)₂D exposure combined with hnRNPC1/C2 knockdown (KD versus 1,25+KD); (D) 1,25(OH)₂D-treated cells compared to 1,25(OH)₂D treatment combined with hnRNPC1/C2 knockdown ((1,25 versus 1,25+KD); and (E) combination of 1,25(OH)₂D treatment and hnRNPC1/C2 knockdown relative to control cells (Ctrl versus 1,25+KD). Absolute numbers of DEG and DSG shown within each segment.