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Molecular Biology Reports

, Volume 46, Issue 6, pp 6243–6252 | Cite as

Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model

  • Caroline Y. Sung
  • Yen-Ting Liu
  • Lynda B. Bennett
  • Caitlin C. Devitt
  • Stephen X. SkapekEmail author
Original Article

Abstract

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins—p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor—and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye—a process essential for normal eye development—does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

Keywords

Arf Ink4a Cis-regulation BAC transgenic mice Eye development 

Notes

Acknowledgements

The authors gratefully acknowledge technical assistance provided by S. Singleterry, and many helpful comments by other members of the Skapek laboratory. We also acknowledge support from the National Institutes of Health National Eye Institute (Grant No. R01 EY019942), from the Cancer Prevention and Research Institute of Texas (Grant No. RP120685-P2), and from the UTSW Harold C. Simmons Comprehensive Cancer Center from the National Cancer Institute (Grant No. CA142543).

Compliance with ethical standards

Conflict of interest

We have no conflict of interest related to this report.

Supplementary material

11033_2019_5063_MOESM1_ESM.docx (497 kb)
Supplementary material 1 (DOCX 496 kb)

References

  1. 1.
    Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, Johnson BE, Skolnick MH (1994) A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436–440.  https://doi.org/10.1126/science.8153634 CrossRefPubMedGoogle Scholar
  2. 2.
    Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368:753–756.  https://doi.org/10.1038/368753a0 CrossRefPubMedGoogle Scholar
  3. 3.
    Quelle DE, Zindy F, Ashmun RA, Sherr CJ (1995) Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83:993–1000.  https://doi.org/10.1016/0092-8674(95)90214-7 CrossRefPubMedGoogle Scholar
  4. 4.
    Gil J, Peters G (2006) Regulation of the INK4bARFINK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677.  https://doi.org/10.1038/nrm1987 CrossRefPubMedGoogle Scholar
  5. 5.
    Somasundaram K, EI-Deiry WS (2000) Tumor suppressor p53: regulation and function. Front Biosci 5:D424–D437.  https://doi.org/10.2741/Somasund CrossRefPubMedGoogle Scholar
  6. 6.
    Silva RL, Thornton JD, Martin AC, Rehg JE, Bertwistle D, Zindy F, Skapek SX (2005) Arf-dependent regulation of Pdgf signaling in perivascular cells in the developing mouse eye. EMBO J 24:2803–2814.  https://doi.org/10.1038/sj.emboj.7600751 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bringold F, Serrano M (2000) Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol 35:317–329.  https://doi.org/10.1016/S0531-5565(00)00083-8 CrossRefPubMedGoogle Scholar
  8. 8.
    Sherr CJ (2012) Ink4-Arf locus in cancer and aging. Wiley Interdiscip Rev Dev Biol 1:731–741.  https://doi.org/10.1002/wdev.40 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kim WY, Sharpless NE (2006) The regulation of INK4/ARF in cancer and aging. Cell 127:265–275.  https://doi.org/10.1016/j.cell.2006.10.003 CrossRefPubMedGoogle Scholar
  10. 10.
    Almog N, Rotter V (1997) Involvement of p53 in cell differentiation and development. Biochim Biophys Acta 1333:F1–F27.  https://doi.org/10.1016/S0304-419X(97)00012-7 CrossRefPubMedGoogle Scholar
  11. 11.
    Lipinski MM, Jacks T (1999) The retinoblastoma gene family in differentiation and development. Oncogene 18:7873–7882.  https://doi.org/10.1038/sj.onc.1203244 CrossRefPubMedGoogle Scholar
  12. 12.
    Stiewe T (2007) The p53 family in differentiation and tumorigenesis. Nat Rev Cancer 7:165–167.  https://doi.org/10.1038/nrc2072 CrossRefPubMedGoogle Scholar
  13. 13.
    Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, Ashmun RA, Grosveld G, Sherr CJ (1997) Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19 ARF. Cell 91:649–659.  https://doi.org/10.1016/S0092-8674(00)80452-3 CrossRefPubMedGoogle Scholar
  14. 14.
    Serrano M, Lee H-W, Chin L, Cordon-Cardo C, Beach D, DePinho RA (1996) Role of the INK4a locus in tumor suppression and cell mortality. Cell 85:27–37.  https://doi.org/10.1016/S0092-8674(00)81079-X CrossRefPubMedGoogle Scholar
  15. 15.
    Sharpless NE, Bardeesy N, Lee K-H, Carrasco D, Castrillon DH, Aguirre AJ, Wu EA, Horner JW, DePinho RA (2001) Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413:86–91.  https://doi.org/10.1038/35092592 CrossRefPubMedGoogle Scholar
  16. 16.
    McKeller RN, Fowler JL, Cunningham JJ, Warner N, Smeyne RJ, Zindy F, Skapek SX (2002) The Arf tumor suppressor gene promotes hyaloid vascular regression during mouse eye development. Proc Natl Acad Sci USA 99:3848–3853.  https://doi.org/10.1073/pnas.052484199 CrossRefPubMedGoogle Scholar
  17. 17.
    Churchman ML, Roig I, Jasin M, Keeney S, Sherr CJ (2011) Expression of Arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells. PLoS Genet 7:e1002157.  https://doi.org/10.1371/journal.pgen.1002157 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Freeman-Anderson NE, Zheng Y, McCalla-Martin AC, Treanor LM, Zhao YD, Garfin PM, He T-C, Mary MN, Thornton JD, Anderson C, Gibbons M, Saab R, Baumer SH, Cunningham JM, Skapek SX (2009) Expression of the Arf tumor suppressor gene is controlled by TGFβ2 during development. Development 136:2081–2089.  https://doi.org/10.1242/dev.033548 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Zheng Y, Zhao YD, Gibbons M, Abramova T, Chu PY, Ash JD, Cunningham JM, Skapek SX (2010) Tgfβ signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK. J Biol Chem 285:35654–35664.  https://doi.org/10.1074/jbc.M110.128959 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McPherson R, Pertsemlidis A, Kavaslar N, Stewart A, Roberts R, Cox DR, Hinds DA, Pennacchio LA, Tybjaerg-Hansen A, Folsom AR, Boerwinkle E, Hobbs HH, Cohen JC (2007) A common allele on chromosome 9 associated with coronary heart disease. Science 316:1488–1491.  https://doi.org/10.1126/science.1142447 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hannou SA, Wouters K, Paumelle R, Staels B (2015) Functional genomics of the CDKN2A/B locus in cardiovascular and metabolic disease: what have we learned from GWASs? Trends Endocrinol Metab 26:176–184.  https://doi.org/10.1016/j.tem.2015.01.008 CrossRefPubMedGoogle Scholar
  22. 22.
    Visel A, Zhu Y, May D, Afzal V, Gong E, Attanasio C, Blow MJ, Cohen JC, Rubin EM, Pennacchio LA (2010) Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464:409–412.  https://doi.org/10.1038/nature08801 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zheng Y, Devitt C, Liu J, Mei J, Skapek SX (2013) A distant, cis-acting enhancer drives induction of Arf by Tgfβ in the developing eye. Dev Biol 380:49–57.  https://doi.org/10.1016/j.ydbio.2013.05.003 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Esteller M (2002) CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 21:5427–5440.  https://doi.org/10.1038/sj.onc.1205600 CrossRefPubMedGoogle Scholar
  25. 25.
    Bernard D, Martinez-Leal JF, Rizzo S, Martinez D, Hudson D, Visakorpi T, Peters G, Carnero A, Beach D, Gil J (2005) CBX7 controls the growth of normal and tumor-derived prostate cells by repressing the Ink4a/Arf locus. Oncogene 24:5543–5551.  https://doi.org/10.1038/sj.onc.1208735 CrossRefPubMedGoogle Scholar
  26. 26.
    Chen H, Gu X, Su I-h, Bottino R, Contreras JL, Tarakhovsky A, Kim SK (2009) Polycomb protein Ezh2 regulates pancreatic β-cell Ink4a/Arf expression and regeneration in diabetes mellitus. Genes Dev 23:975–985.  https://doi.org/10.1101/gad.1742509 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M (1999) The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397:164–168.  https://doi.org/10.1038/16476 CrossRefPubMedGoogle Scholar
  28. 28.
    Martin AC, Thornton JD, Liu J, Wang X, Zuo J, Jablonski MM, Chaum E, Zindy F, Skapek SX (2004) Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the Arf tumor suppressor gene. Investig Ophthalmol Vis Sci 45:3387–3396.  https://doi.org/10.1167/iovs.04-0349 CrossRefGoogle Scholar
  29. 29.
    Zindy F, Quelle DE, Roussel MF, Sherr CJ (1997) Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 15:203–211.  https://doi.org/10.1038/sj.onc.1201178 CrossRefPubMedGoogle Scholar
  30. 30.
    Young NP, Jacks T (2010) Tissue-specific p19Arf regulation dictates the response to oncogenic K-ras. Proc Natl Acad Sci USA 107:10184–10189.  https://doi.org/10.1073/pnas.1004796107 CrossRefPubMedGoogle Scholar
  31. 31.
    Zindy F, Williams RT, Baudino TA, Rehg JE, Skapek SX, Cleveland JL, Roussel MF, Sherr CJ (2003) Arf tumor suppressor promoter monitors latent oncogenic signals in vivo. Proc Natl Acad Sci USA 100:15930–15935.  https://doi.org/10.1073/pnas.2536808100 CrossRefPubMedGoogle Scholar
  32. 32.
    Burd CE, Sorrentino JA, Clark KS, Darr DB, Krishnamurthy J, Deal AM, Bardeesy N, Castrillon DH, Beach DH, Sharpless NE (2013) Monitoring tumorigenesis and senescence in vivo with a p16INK4a-luciferase model. Cell 152:340–351.  https://doi.org/10.1016/j.cell.2012.12.010 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  34. 34.
    Sharpless NE (2006) Chapter 28—preparation and immortalization of primary murine cells. In: Celis JE (ed) Cell biology, 3rd edn. Academic Press, Burlington, pp 223–228.  https://doi.org/10.1016/B978-012164730-8/50029-0 CrossRefGoogle Scholar
  35. 35.
    Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17:299–313.  https://doi.org/10.1083/jcb.17.2.299 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Gong S, Yang XW, Li C, Heintz N (2002) Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6kγ origin of replication. Genome Res 12:1992–1998.  https://doi.org/10.1101/gr.476202 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Urasaki A, Morvan G, Kawakami K (2006) Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174:639–649.  https://doi.org/10.1534/genetics.106.060244 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Datta S, Costantino N, Court DL (2006) A set of recombineering plasmids for gram-negative bacteria. Gene 379:109–115.  https://doi.org/10.1016/j.gene.2006.04.018 CrossRefPubMedGoogle Scholar
  39. 39.
    Suster ML, Sumiyama K, Kawakami K (2009) Transposon-mediated BAC transgenesis in zebrafish and mice. BMC Genom 10:477.  https://doi.org/10.1186/1471-2164-10-477 CrossRefGoogle Scholar
  40. 40.
    Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL (2009) Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res 18:769–785.  https://doi.org/10.1007/s11248-009-9271-2 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Suster ML, Abe G, Schouw A, Kawakami K (2011) Transposon-mediated BAC transgenesis in zebrafish. Nat Protoc 6:1998–2021.  https://doi.org/10.1038/nprot.2011.416 CrossRefPubMedGoogle Scholar
  42. 42.
    Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73:2886–2892PubMedPubMedCentralGoogle Scholar
  43. 43.
    Liu Y-T, Xu L, Bennett L, Hooks JC, Liu J, Zhou Q, Liem P, Zheng Y, Skapek SX (2019) Identification of de novo enhancers activated by TGFβ to drive expression of CDKN2A and B in HeLa cells. Mol Cancer Res.  https://doi.org/10.1158/1541-7786.Mcr-19-0289 CrossRefPubMedGoogle Scholar
  44. 44.
    Kristensen DM, Wolf YI, Mushegian AR, Koonin EV (2011) Computational methods for gene orthology inference. Brief Bioinform 12:379–391.  https://doi.org/10.1093/bib/bbr030 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Matheu A, Pantoja C, Efeyan A, Criado LM, Martín-Caballero J, Flores JM, Klatt P, Serrano M (2004) Increased gene dosage of Ink4a/Arf results in cancer resistance and normal aging. Genes Dev 18:2736–2746.  https://doi.org/10.1101/gad.310304 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Narayanan K, Chen Q (2011) Bacterial artificial chromosome mutagenesis using recombineering. J Biomed Biotechnol 2011:971296.  https://doi.org/10.1155/2011/971296 CrossRefPubMedGoogle Scholar
  47. 47.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278.  https://doi.org/10.1016/j.cell.2014.05.010 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Yang H, Wang H, Jaenisch R (2014) Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc 9:1956–1968.  https://doi.org/10.1038/nprot.2014.134 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Caroline Y. Sung
    • 1
  • Yen-Ting Liu
    • 1
  • Lynda B. Bennett
    • 1
  • Caitlin C. Devitt
    • 1
  • Stephen X. Skapek
    • 1
    • 2
    Email author
  1. 1.Division of Hematology/Oncology, Department of PediatricsUT Southwestern Medical CenterDallasUSA
  2. 2.Harold C. Simmons Comprehensive Cancer CenterUniversity of Texas Southwestern Medical CenterDallasUSA

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