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Splicing factor SRSF6 promotes hyperplasia of sensitized skin


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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Figure 1: SRSF6 overexpression induces skin and intestinal hyperplasia in mice.
Figure 2: SRSF6 overexpression results in a wound-healing expression signature.
Figure 3: SRSF6 is involved in wound healing.
Figure 4: SRSF6 regulates AS in skin hyperplasia.
Figure 5: Tenascin-C AS is regulated by SRSF6 in skin.
Figure 6: Tnc-FL expression is linked to hyperplasia severity.
Figure 7: SRSF6 is a proto-oncogene overexpressed in human skin cancer.
Figure 8: Model for the dynamic role of SRSF6 in skin hyperplasia and wound healing.

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We thank R. Jaenisch (Whitehead Institute) for KH2 ES cells; C. Miething and S.W. Lowe (Memorial Sloan-Kettering Cancer Center) for the targeting construct; P. Premsrirut for useful discussions and help with production of stable embryonic stem-cell lines; S.Y. Kim from the Gene Targeting Shared Resource (Cold Spring Harbor Laboratory) for help with tetraploid complementation procedures; C. Johns for support with microarray analysis; M. Motley for assistance with RT-PCR analysis; and L. Chartarifsky for tissue-microarray staining. We thank L. Bianco for help with animal procedures. We are grateful to B. Boettner, M. Egeblad and A. Mills for critical comments on the manuscript. This study was supported by grant CA13107 from the US National Cancer Institute and was performed with assistance from Cold Spring Harbor Laboratory Shared Resources funded in part by Cancer Center Support grant 5P30CA045508. M.A.J. was supported by a Danish Cancer Society postdoctoral fellowship.

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Authors and Affiliations



M.A.J. and A.R.K. designed the study and wrote the paper. M.A.J. and J.E.W. carried out the experiments and analyzed the data. All authors read the manuscript.

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Correspondence to Adrian R Krainer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Tet-on transgenic SRSF6 mouse.

(a) A human SRSF6 cDNA (with an N-terminal T7-tag) was inserted into pBS31'-RBGpA-TREtight-ColA1 flp-in. Upon induction, the cDNA is expressed as a bicistronic transcript, including an IRES element followed by EGFP cDNA. The reverse tetracycline-controlled transactivator (M2-rtTA) is expressed from the Rosa26 locus in KH2 ES cells. (b) Expression of ectopic SRSF6 (T7) and GFP protein was measured by immunoblotting using antibodies against the T7-tag or GFP upon DOX treatment of R26-rtTA/ColA1-SRSF6-transgenic mice. Protein was extracted from thymus (th), liver (li), skin (sk), small intestine (in), brain (br), heart (he), kidney (ki), and spleen (sp). Loading: 0.1% Ponceau S staining. (c) Sporadic expression of ectopic SRSF6 in bone marrow. Control (left panel), +DOX (right panel). T7-tag antibody detects ectopically expressed SRSF6. Bar = 100 μm (upper panel), 20 μm (lower panel).

Supplementary Figure 2 Epidermal hyperplasia in mild to moderate lesions.

(a) Epidermis from transgenic mouse not exposed to doxycycline. Note continuous basal layer, 1-2 cell thick spinous layer, and thin stratum corneum (bar = 50 μm). (b) Normal epidermis 200 μm from area of hyperplasia (bar = 50 μm). (c) Overexpression of SRSF6 for 7 days leads to mild epidermal hyperplasia. Note the orthokeratotic hyperkeratotic (arrow) stratum corneum. The granular layer is also more prominent (short arrow) (bar = 50 μm). (d) Prominent thickened stratum spinosum (arrow) and minimal dysplasia in mild early lesion (bar = 50 μm). (e) Overexpression of SRSF6 for 14 days results in severe hyperplasia. Note the severe hyperplasia in the epidermis and hair follicles and thick parakeratotic hyperkeratotic crust (arrow) (bar = 100 μm). (f) Higher magnification of e. There is severe hyperplasia of the epidermis (arrow) and hair follicles (short arrow) (bar = 100 μm).

Supplementary Figure 3 Abnormalities in the epidermis after overexpression of SRSF6 for 14–21 d.

(a) Intra-epidermal microabscess (short arrow) and mild inflammation (arrow) at the disturbed dermal-epidermal interface. (bar = 100 μm). (b) Multiple apoptotic bodies (arrows) and dyskeratosis (faulty development of the epidermis with abnormal keratinization) (short arrow) (bar = 100 μm). (c) Spongiosis (loss of adhesion and intercellular edema) in the epidermis (bar = 100 μm). (d) Basal cell apoptosis (Civatte body) (arrow), spongiosis, and dyskeratosis (bar = 100 μm).

Supplementary Figure 4 Verrucose hyperplasia in plucked skin of mice overexpressing SRSF6 for 21 d.

(a) Severe epidermal hyperplasia (short arrow) and exorbitant parakeratotic hyperkeratosis (arrow) (bar = 100 μm). (b) Dyskeratosis with severe parakeratosis (arrows). Hair follicles (short arrows) are trapped in the rapidly proliferating hyperplastic epidermis (bar = 50 μm). (c) Thickened epidermis (dotted line) with extensive hyperkeratosis and keratin pearls trapped in the hyperplastic stratum corneum (arrow) (bar = 50 μm). (d) Severe dysplasia with severe dyskeratosis, excessive apoptotic bodies (arrows), and dysplastic keratinocytes (short arrows) (bar = 25 μm).

Supplementary Figure 5 RT-PCR validation of differentially expressed genes upon SRSF6 overexpression in skin.

14 out of 14 genes tested were successfully validated by RT-PCR. Confirmed upregulated genes include: genes induced upon tissue injury, such as Keratin 6 (Krt6) (microarray: +154-fold), Keratin 16 (Krt16) (microarray: +13-fold) and tenascin C (Tnc) (microarray: +6-fold); and pro-inflammatory cytokines important for wound healing, such as tumor necrosis factor (Tnf) (microarray: +6-fold), Interleukin-1 b (Il1b) (microarray: +24-fold), Chemokine (C-X-C motif) ligand 2 (Cxcl2) (microarray: +97-fold), Chemokine (C-C motif) ligand 3 (Ccl3) (microarray: +43-fold). Confirmed downregulated genes included: genes related to skin stem cells, such as Keratin 15 (Krt15) (microarray: -5-fold), Leucine-rich repeat-containing G-protein coupled receptor 6 (Lgr6) (microarray: -5-fold); and genes related to differentiation, such as Filaggrin-2 (Flg2) (microarray: -4-fold) and Stearoyl-coenzyme A desaturase 3 (Scd3) (microarray: -18-fold).

Supplementary Figure 6 In vitro differentiation of primary keratinocytes.

Primary keratinocytes from control (R26-rtTA) or double-transgenic (R26-rtTA/ColA1-SRSF6) mice were allowed to differentiate in the presence of 1.2 mM Ca2+ for 5 d. (a) Induction of SRSF6 by DOX-treatment (+) strongly promoted differentiation of primary compared to control keratinocytes, as seen by the strong induction of genes associated with keratinocyte differentiation, measured by RT-qPCR: loricrin (Lor: +4-fold), involucrin (Inv: +7-fold), keratin 1 (Krt1: +743-fold), filaggrin (Flg: +32-fold). In contrast, genes associated with undifferentiated keratinocytes, such as keratin 5 (Krt5: +3-fold) and keratin 14 (Krt14: +2-fold) were relatively unchanged upon SRSF6 induction. (b) The corresponding increase in loricrin protein levels upon SRSF6 induction was validated by immunoblotting; no change was observed in DOX-treated control keratinocytes (note: loading was different than for double-transgenic keratinocytes). Keratin 5 protein levels were unaffected by DOX treatment in both control and double-transgenic keratinocytes.

Supplementary Figure 7 Splicing-target validation.

Validation of SRSF6-responsive ASEs using 32P-radioactive RT-PCR. (a) Strong ASEs with significant change; *p<0.05: Phosphatidylinositol transfer protein, cytoplasmic 1 (Pitpnc1), kinesin family member 20B (Kif20b), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (Magi1), synaptotagmin VIII (Syt8), hexokinase 3 (Hk3), SH2-domain-containing 5 (Sh2d5), lines homolog (Drosophila)(Lins2), ankyrin 1, erythrocytic (Ank1), ETS-domain protein (SRF accessory protein 1) (Elk4), Pyruvate Kinase 2 (Pkm2). (b) Weak ASEs that were not significant: tryptase alpha/beta (Tpsab1), growth factor receptor-bound protein 7 (Grb7). (c) ASEs that did not show anticipated splicing changes by RT-PCR and therefore could not be validated: brain-specific angiogenesis inhibitor 2 (Bai2), valosin containing protein (Vcp), family with sequence similarity 160, member B2 (Fam160b2), armadillo-repeat gene deleted in velocardiofacial syndrome (Arvcf). Statistical analysis employed a Mann-Whitney test (n=10). Data are represented as mean +/- s.e.m. (d) Tnc splicing changes upon Srsf6 knockdown in NIH-3T3 cells. 32P-radioactive RT-PCR validation shows reciprocal splicing changes in Tnc upon Srsf6 knockdown, using primers specific for Tnc exons 9 and 16. Tnc +E10-15 and ΔE14 isoforms decreased upon Srsf6 depletion, whereas Tnc-S (ΔE10-15) (constitutive isoform) was unaffected. Lanes shown correspond to two biological replicates. (e) Pictogram of the predicted SRSF6 in vivo binding motif (9-mer; CCWKSWGSM, Top) which shows similarity to the reported SRSF6 functional SELEX binding motif (6-mer; YRCRKM, Bottom). The central 5-mer of the predicted SRSF6 in vivo binding motif is denoted ‘5-mer core motif’.

Supplementary Figure 8 SRSF6 and its target TNC are required for wound healing in vitro.

(a) Human A2058 melanoma cells transduced with inducible short-hairpins against either TNC or SRSF6 were used for wound-healing assays. Cells were treated with DOX (10 μg/ml) for 4 d in total. Cell migration was measured over 18 h. Knockdown of either SRSF6 or TNC impairs wound healing (2 shRNAs per gene). (b) Immunoblotting confirms efficient SRSF6 knockdown. NSC=no-silencing control.

Supplementary Figure 9 Original images.

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Supplementary Figures 1–9, Supplementary Tables 1, 3 and 4, and Supplementary Note (PDF 906 kb)

Supplementary Table 2

Gene expression analysis, functional gene annotation analysis and splicing targets (XLSX 216 kb)

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Jensen, M., Wilkinson, J. & Krainer, A. Splicing factor SRSF6 promotes hyperplasia of sensitized skin. Nat Struct Mol Biol 21, 189–197 (2014).

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