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. 2014 Feb;21(2):189-97.
doi: 10.1038/nsmb.2756. Epub 2014 Jan 19.

Splicing factor SRSF6 promotes hyperplasia of sensitized skin

Mads A Jensen  1 , John E Wilkinson  2 , Adrian R Krainer  3
Affiliations

Splicing factor SRSF6 promotes hyperplasia of sensitized skin

Mads A Jensen et al. Nat Struct Mol Biol. 2014 Feb.

Abstract

Many biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 SRSF6-target genes in skin and show that this SR-rich protein binds to alternative exons in the pre-mRNA of the extracellular-matrix protein tenascin C, thus promoting the expression of isoforms characteristic of invasive and metastatic cancer independently of cell type. SRSF6 overexpression additionally results in depletion of LGR6+ stem cells and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the tenascin-C isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis.

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Figures

Figure 1
Figure 1
SRSF6 overexpression induces skin and intestinal hyperplasia in mice. (a) RT-PCR showing expression of transgenic (tg) and total SRSF6 in DOX-treated R26-rtTA/ColA1-SRSF6-transgenic mice, and Actb mRNA as a loading control, in RNA extracted from thymus (th), liver (li), skin (sk), small intestine (in), brain (br), heart (he), kidney (ki), spleen (sp), and parental SRSF6-targeted ES cells. (b) In vivo imaging showing DOX induction of GFP after 0, 3, 14, or 21 d, in transgenic versus control mice. (c) Left panel: Abnormal skin phenotype of transgenic R26-rtTA/ColA1 mouse treated for 21 d with DOX. Right panel: Magnification of region with abnormal back skin. Bar = 0.5 cm. (d) Analysis of epidermal thickness: control skin (solid square) and SRSF6-expressing skin (solid circle), with thickness in μm (Mann-Whitney test, p<0.0001, skin samples from 3 treated and 3 control mice, n=21 each). Data are represented as mean +/− s.e.m. (e) Histopathology (H&E staining) of transgenic tissues from skin and small intestine of mice induced with DOX for 14 d, showing severe hyperplasia.
Figure 2
Figure 2
SRSF6 overexpression results in a wound-healing expression signature. (a) Two-color immunofluorescence of transgenic SRSF6 (T7), keratin 14 (KRT14), keratin 6 (KRT6) and KI67 in transgenic skin (DOX: +SRSF6). (b) Left panel: nude mouse with transplanted skin graft, 1 month after surgery. Middle panel: bright field (BF) or GFP images of allogenic skin transplants induced by DOX for 1 week (Control versus R26-rtTA/ColA1-SRSF6). Right panel: Histopathology showing skin hyperplasia in SRSF6-expressing transplants (H&E staining); n=6. (c) Expression of an epidermal differentiation marker, loricrin (LOR), a basal cell marker, keratin 5 (KRT5), and SRSF6. (d) Unsupervised hierarchical clustering of SRSF6-induced versus control skin (n=4) based on the 25 most variable genes; DOX-treated (1+ and 2+) and control (3− and 4−) samples represent biological replicates. (e) Top: Analysis of fibrillar type I+III collagen levels by Picrosirius-red staining of skin obtained from DOX-induced (+SRSF6) or control transgenic mice. Images are for bright field (BF) or polarized light (PL) conditions. Bottom: Image quantification of thin (green), thick (red), or total (gray) collagen (type I and III) (Mann-Whitney test, p<0.0001, n=60 fields per condition). Bar = 50 μm (a, c), 2 mm (b), and 100 μm (e). Data are represented with the mean for each group.
Figure 3
Figure 3
SRSF6 is involved in wound healing. (a) Effect of hair shaving on SRSF6-induced skin hyperplasia. (b) Full-thickness incision wound healing of mouse tails from transgenic mice, induced by DOX or controls (1 week). (c) Measurement of wound-induced hyperplasia upon SRSF6 induction. Left: Representative tail-skin wounds in the presence or absence of SRSF6 induction (1 week post-injury), H&E staining; bar = 100 μm. Right: Quantification of epidermal thickness at sites proximal (center) or distal (125 μm from the center) from the wounds (Mann-Whitney test, p<0.001, n=26). (d) Immunoblotting for endogenous SRSF6 expression in wounded or shaved skin from DOX-induced or control mice. Arrow indicates fully phosphorylated SRSF6. (e) qPCR of stem-cell markers in total skin extracts, showing depletion of skin stem cells upon SRSF6-induced hyperplasia: Keratin 15 (Krt15), Lgr4, Lgr5, and Lgr6 (Mann-Whitney test, n=4 samples per condition). (f) Clonogenic assay of primary keratinocytes isolated from R26-rtTA/ColA1-SRSF6 (double-transgenic) or R26-rtTA (control) mice Representative images of Crystal-Violet-stained colonies, showing that SRSF6 overexpression strongly reduced colony formation (plating efficiency %: Fisher’s exact test, p<0.05, n=3 per condition). Data are represented as mean +/− s.e.m.
Figure 4
Figure 4
SRSF6 regulates AS in skin hyperplasia. (a) Microarray analysis of AS events (ASEs) showing 154 changes identified by two independent pairwise comparisons between SRSF6-induced and control skin samples. (b-d) Radioactive RT-PCR validations of SRSF6-responsive ASEs: tenascin C (Tnc), pyruvate kinase 2 (Pkm2), and ETS-domain protein (SRF accessory protein 1) (Elk4) (Mann-Whitney test; *p<0.05, n=10). Data are represented as mean +/− s.e.m.
Figure 5
Figure 5
Tenascin C AS is regulated by SRSF6 in skin. (a) Top: schematic representation of exons 9 to 16 of the mouse Tnc gene. Black: alternative cassette exons (exons 10 to 15) regulated by SRSF6; gray: constitutive exons 9 and 16. Middle: the exon 9 to 16 region of Tnc is highly conserved in vertebrates. Bottom: inverse correlation between putative SRSF6 in vivo binding motif (5-mer core motif; WKSWG) frequency and 5′-splice-site-strength predictions. (b) RNA CLIP from NIH-3T3 cells with primer pairs complementary to mouse Tnc exons 9 and 10, or 15 and 16, respectively. (c) RNA CLIP-qPCR analysis with MNase digestion. (d) RNA-affinity pulldown using immobilized 21-mer RNA oligonucleotides comprising the wild-type SRSF6 binding motif in exon 12 (E12; UGCAGGA), exon 15 (E15; UUCUAGUUUCAGAU) or a mutated motif (mutE12; CAACAUG). (e) Left: qPCR analysis of siRNA knockdown of SRSF6 and luciferase control, showing effects on Tnc AS (Mann-Whitney test, p<0.05, n=6). Right: immunoblotting after SRSF6 knockdown showing effect on TNC protein isoform levels. Data are represented as mean +/− s.e.m.
Figure 6
Figure 6
Tnc-FL expression is linked to hyperplasia severity. (a) qPCR analysis of skin from time-course experiment. Left: total SRSF6; right: Tnc isoform ratio (Tnc-FL/Total Tnc) (n=3). (b) Immunofluorescence of TNC-FL protein isoform expression in skin upon SRSF6 induction for 0 h and 7 d. Epidermis=epi. (c) Correlation between skin hyperplasia, SRSF6 induction, and splicing switch towards the Tnc-FL isoform; 0 h to 7 d. Bottom right: Reversion of skin hyperplasia upon DOX withdrawal (5 d) after SRSF6 induction (2 d). (d) In vitro wound-healing assay (18 h), showing enhancement of cell migration by SRSF6 overexpression in NIH-3T3 cells (n=24, p<0.0001) (left), and qPCR analysis of concomitant increases in relative Tnc-FL mRNA levels (right). (e) Cell migration assay showing that shRNA knockdown of Tnc suppresses the effect of SRSF6 overexpression (n=24, p<0.0001) (left); qPCR analysis of relative Tnc-FL mRNA levels (right). (f) In vitro wound-healing assay; inducible shRNA knockdown of either TNC or SRSF6 (two hairpins each) in A2058 human melanoma cells, showing reduced cell migration; +DOX (10 μg/ml) for 4 d (n=24, p<0.0001) (left). qPCR analysis of relative TNC-FL mRNA levels after knockdown of TNC or SRSF6 (right). Bar = 100 μm (b, c). Data are represented as mean +/− s.e.m. All p-values were calculated by Mann-Whitney tests.
Figure 7
Figure 7
SRSF6 is a proto-oncogene overexpressed in human skin cancer. (a) Analysis of tumor growth in nude mice injected with 2×106 MEF +c-Myc, p53−/− cells in each flank (n=8). Control (A), SRSF6 (B). (b) Tissue microarray analysis of SRSF6 protein expression in human tumors, showing SRSF6 overexpression (High SRSF6) in BCC (100%, p<0.0001, n=20), SCC (100%, p<0.0001, n=18), and malignant melanoma (52%, p=0.01, n=17), compared to normal skin (10%, n=20). (c) Serial tissue microarrays of malignant melanoma tissues stained with either anti-SRSF6 or anti-TNC-FL antibodies for expression-correlation analysis (n=92). Tumor #1-2: representative tissues. (d) Diagram showing strong correlation between levels of SRSF6 and TNC-FL proteins in most of the tissues analyzed (66.3%). Data in a are represented as mean +/− s.e.m. All p-values were calculated by Fisher’s exact test.
Figure 8
Figure 8
Model for the dynamic role of SRSF6 in skin hyperplasia and wound healing. Bottom (blue): Normal tissue homeostasis in skin. Top (red): During tissue injury, expression of SRSF6 is induced (and perhaps required) to regulate or promote unknown processes that are part of normal wound healing, through AS of target genes, e.g., Tnc and others. When SRSF6 expression is elevated, skin stem cells are aberrantly activated upon stimulation (by shaving or wounding) causing skin hyperplasia. SRFS6 likely suppresses stem-cell self-renewal signals and/or stimulates stem-cell differentiation to keratinocyte progenitors that are maintained in a proliferating state. Aberrant levels of the SRSF6 splicing target Tnc-FL protein isoform may have a disruptive role for maintenance of the stem-cell niche microenvironment.
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