Fig 1.
Identification of SMCs in the intestinal smooth muscle with eGFP and MYH11 antibody.
(A) A confocal microscopy z-stack image of whole-mount jejunum muscularis showing longitudinal and circular SMCs expressing eGFP. (B) Immunohistochemistry of SMCs using anti-MYH11(smMHC) antibody. (C) Merged images of eGFP and MYH11(smMHC). (D & E) Primary eGFP+ SMCs from jejunum and colon identified (circled) on flow cytometry.
Fig 2.
Comparison of transcriptomes obtained from jejunal and colonic SMCs.
(A) Venn diagram showing the number of genes expressed in JSMCs, CSMCs, jejununal muscularis, and colonic muscularis. (B) Comparison of expression levels of genes in JSMCs and CSMCs. (C) Comparison of correlation coefficients between JSMCs, CSMCs, jejununal muscularis, and colonic muscularis. (D) Gene ontologies reported in JSMCs and CSMCs. Gene ontology (GO: function, process, and component) of SMC-specific genes was analyzed, and key GO terms were compared with a normalized expression percentile.
Fig 3.
SMC transcriptome, SRF binding sites, and CArG boxes built on the UCSC genome browser.
(A) A genomic map view of CArG boxes and SRF binding sites on Acta2, a gene expressed in SMCs. All predicted CArG boxes were mapped on the genome. Conserved CArG boxes beween mice and humans are indicated by red circles. Four transcriptional variants with alternative initiation sites were expressed in colonic and jejunal SMCs (set by view options; large red horizontal arrows). The transcriptional variants were aligned with H3K27ac sites (small intestine), RNA polymerase II and SRF binding sites (C2C12 myoblasts), which were publically available in the UCSC database. The two SRF binding sites contained three CArG boxes (red circles), which were located in the promoter and intron 1 regions, and conserved beween mice and humans (set by view options). Note that the SRF binding sites aligned with that of RNA polymerase II and the H3K27ac sites from SRF ChIP data. Directionality of each mRNA on the browser is indicated by the arrows on a line (e.g., >>>, sense strand; <<<, antisense strand). (B) Expression levels (FPKM) of Acta2 in SMCs and whole tissue. (C) Expression levels (FPKM) of Acta2 transcriptional variants in SMCs. The variant (V) ID represents the last three digits of the TCONS number. (D) PCR validation of Acta2 exons with different transcriptional initiation sites in isolated SMCs and muscularis of jejunum and colon. NTC is non-template control. Primer sets were designed from variant exons in the regions of interest (see S11 Table for primer sequences).
Fig 4.
Analysis of CArG boxes and SRF binding sites on SMC transcriptome.
A total of 1,540 SRF binding sites (peak height >1.5) obtained from SRF ChIP-seq of C2C12 myoblasts were analyzed for association with CArG boxes, CpG islands, and genes. (A) Proportion of SRF binding sites associated with CArG boxes. (B) Proportion of SRF binding sites associated with CArG boxes conserved between mice and humans. (C) Proportion of SRF binding sites associated with CpG islands. (D) Proportion of SRF binding sites associated with genes. (E) Locations of SRF binding sites on genes expressed in SMCs. (F) A representative list of SMC genes that were highly expressed and associated with SRF binding sites. Eighty-six genes containing SRF binding sites (peak hight >1.5) and highly expressed (>100 FPKM) in SMCs are shown. The SMC genes of the 34 proteins regulated by SRF in SMC-specific Srf KO mice are indicated in bold green letters. (G) Proportion of SRF-regulated proteins identified in jejunal SM from SMC-specific Srf KO mice. Eight-six SRF-associated genes were compared with the SRF-regulated proteins identified in a proteomics study involving Srf KO mice: 34 proteins were down-regulated (SRF-regulated; blue), 40 proteins were not detected (red), and 12 proteins did not change expression levels (others; green) in the Srf KO SM.
Fig 5.
Identification of SMC-specific genes by expression profiles and histone modifications.
(A) Specificity of SMC-enriched genes. Cell specificity was determined by comparative analysis of gene expression profiles among SMCs, ICCs, and PDGFRα+ cells: SMCsexpression level (FPKM)/[ICCexpression level (FPKM) + PDGFRα+ cellsexpression level (FPKM)] (B) Comparison of JSMC- and CSMC-enriched gene expression. (C) Genomic map views of histone modications (H3K4m3, H3K27a, and H3K27m3 in 8 week small intestine) on Cnn1 (specifically expressed in JSMCs and CSMCs) and Ubb (ubiquitiously expressed). (D) Comparison of histone modifications on SMC-enriched genes. The signal value is the average of the mininum and maxium values of the ChIP-seq signal for each histone modification. (E) Comparison of histone modifications on ubiquitious genes. (F) Expression levels of SMC-enriched genes not altered by histone modifications.
Fig 6.
Comparison of ion channel and transporter isoform genes expressed in SMCs.
(A) Comparison of expression levels of ion channel isoforms in JSMCs and CSMCs. (B) Comparison of expression levels of ion transproter isoforms in JSMCs and CSMCs. (C) Calcium channel isoforms enriched in JSMCs and CSMCs. (D) SMC-specific calcium channel isoforms. Cell specificity was determined by comparative analysis of gene expression profiles among SMCs, ICC, and PDGFRα+ cells. (E) Voltage-dependent calcium channel isoforms (L-type, Cacna1c & d; T-type, Cacna1h & g; R-type, Cacna1e; P/Q-type, Cacna11) expressed in JSMCs. (F) Voltage-dedendent calcium channel isoforms in CSMCs.
Fig 7.
Identification of an SMC-specific Ryr3 regulated by SRF.
(A) A genomic map view of Ryr3 variants expressed in JSMCs and CSMCs. Three variants TCONS_00167498 (V498, blue), TCONS_00152671 (V671, green), and TCONS_00166589 (V589, red) are indicated by arrows. (B) Expression levels of total Ryr3 in SMCs. (C) Expression levels of each Ryr3 vaiant in SMCs. Variants are arranged on the X-axis from longest (left) to shortest (right) in length. The three variants shown on A are also indicated by the color arrows. (D) A topological map of RYR3 variants. Each circle denotes the corresponding amino acid. Colors on amino acid sequence show distinct regions and domains: red, missing or inserted peptides from differentially spliced exons; green, start codons found in differentially spliced variants. Seven transmembrane domains 1–7 and a pore region (magenta) are shown. MR1-5 (blue), SPRY1-3 (dark green), FKBP1A (orange), CALM (purple), and a residue E (black) that is required for Ca2+ binding (Ca2+) are also indicated. The topology map was drawn based on the longest peptide variant V498. V671 is a C-terminal truncated variant (V671CT) that is missing Ca2+ binding and transmembrane domains after the CALM domain while V589 is an N-terminal truncated variant (V589NT) that is missing the N-terminal domains (N-ter to CALM domain). (E) Western blot showing that RYR3 protein expression decreased in jejunum SM of inducible SMC-specific Srf KO mice as SRF protein was depleted 5, 10, 15, and 20 days following tamoxifen administration. UBE1 was used as an endogenous control.