Skip to main content

Experimental Strategies to Manipulate the Cellular Levels of the Multifunctional Factor CTCF

  • Protocol
  • First Online:
Cancer Cell Signaling

Abstract

Cellular homeostasis is the result of an intricate and coordinated combinatorial of biochemical and molecular processes. Among them is the control of gene expression in the context of the chromatin structure which is central for cell survival. Interdependent action of transcription factors, cofactors, chromatin remodeling activities, and three-dimensional organization of the genome are responsible to reach exquisite levels of gene expression. Among such transcription factors there is a subset of highly specialized nuclear factors with features resembling master regulators with a large variety of functions. This is turning to be the case of the multifunctional nuclear factor CCCTC-binding protein (CTCF) which is involved in gene regulation, chromatin organization, and three-dimensional conformation of the genome inside the cell nucleus. Technically its study has turned to be challenging, in particular its posttranscriptional interference by small interference RNAs. Here we describe three main strategies to downregulate the overall abundance of CTCF in culture cell lines.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Boudreau RL, Davidson BL (2012) Generation of hairpin-based RNAi vectors for biological and therapeutic applications. Methods Enzymol 507:275–296

    Article  CAS  PubMed  Google Scholar 

  2. Phillips JE, Corces VG (2009) CTCF: master weaver of the genome. Cell 137:1194–1211

    Article  PubMed Central  PubMed  Google Scholar 

  3. Herold M, Bartkuhn M, Renkawitz R (2012) CTCF: insights into insulator function during development. Development 139:1045–1057

    Article  CAS  PubMed  Google Scholar 

  4. Lobanenkov VV, Nicolas RH, Adler VV, Paterson H, Klenova EM, Polotskaja AV et al (1990) A novel sequence-specific DNA binding protein which interacts with three regulatory spaced direct repeats of the CCCTC-motif in the 5′ flanking sequence of the chicken c-myc gene. Oncogene 5:1743–1753

    CAS  PubMed  Google Scholar 

  5. Philippova GN, Qi CF, Ulmer JE, Moore JM, Wards MD, Hu YJ et al (2002) Tumor-associated zinc finger mutations in the CTCF transcription factor selectively alter its DNA-binding specificity. Cancer Res 62:48–52

    Google Scholar 

  6. Ohlsson R, Lobanenkov V, Klenova E (2010) Does CTCF mediate between nuclear organization and gene expression? BioEssays 32:37–50

    Article  CAS  PubMed  Google Scholar 

  7. Valadez-Graham V, Razin SV, Recillas-Targa F (2004) CTCF-dependent enhancer blocker at the upstream region of the chicken α-globin gene domain. Nucleic Acids Res 32:1354–1362

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Engel N, West AG, Felsenfeld G, Bartolomei MS (2004) Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations. Nat Genet 36:883–888

    Article  CAS  PubMed  Google Scholar 

  9. De La Rosa-Velázquez IS, Rincón-Arano H, Benítez-Bribiesca L, Recillas-Targa F (2007) Epigenetic regulation of the human retinoblastoma tumor suppressor gene promoter by CTCF. Cancer Res 67:2577–2585

    Article  Google Scholar 

  10. El-Kady A, Klenova E (2005) Regulation of the transcription factor, CTCF, by phosphorylation with protein kinase CK2. FEBS Lett 579:1424–1434

    Article  CAS  PubMed  Google Scholar 

  11. Docquier F, Jita GX, Farrar D, Jat P, O’Hare M, Chernukhin I et al (2009) Decreased poly(ADP-ribosyl)ation of CTCF, a transcription factor, is associated with breast cancer phenotype and cell proliferation. Clin Cancer Res 15:5762–5771

    Article  CAS  PubMed  Google Scholar 

  12. Kitchen NS, Schoenherr CJ (2010) Sumoylation modulates a domain in CTCF that activates transcription and decondenses chromatin. J Cell Biochem 11:665–675

    Article  Google Scholar 

  13. MacPherson MJ, Beatty LG, Zhoi W, Du M, Sdowski PD (2009) The CTCF insulator protein posttranslationally modified by SUMO. Mol Cell Biol 29:714–725

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Docquier F, Farrar D, D’Arcy V, Chernukhin I, Robinsin AF, Loukinov D et al (2005) Heightened expression of CTCF in breast cancer cells is associated with resistance to apoptosis. Cancer Res 65:5112–5122

    Article  CAS  PubMed  Google Scholar 

  15. Soshnikova N, Montavon T, Leleu M, Galjart N, Duboule D (2010) Functional analysis of CTCF during mammalian limb development. Dev Cell 19:819–830

    Article  CAS  PubMed  Google Scholar 

  16. Delgado-Olguín P, Brand-Arzamendi K, Scott IC, Jungblut B, Stainier DY, Bruneau BG et al (2011) CTCF promotes muscle differentiation by modulating the activity of myogenic regulatory factors. J Biol Chem 286:12483–12484

    Article  PubMed Central  PubMed  Google Scholar 

  17. Liu Z, Scannell DR, Eisen MB, Tjian R (2011) Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146:720–731

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Fiorentino FP, Giordano A (2011) The tumor suppressor role of CTCF. J Cell Physiol 227:479–492

    Article  Google Scholar 

  19. Recillas-Targa F, De La Rosa-Velázquez IA, Soto-Reyes E (2011) Insulation of tumor suppressor genes by the nuclear factor CTCF. Biochem Cell Biol 89:479–488

    Article  PubMed  Google Scholar 

  20. Barkess G, West AG (2012) Chromatin insulator elements: establishing barriers to set heterochromatin boundaries. Epigenomics 4:67–80

    Article  CAS  PubMed  Google Scholar 

  21. Burgess-Beusse B, Farrell C, Gaszner M, Litt M, Mutskov V, Recillas-Targa F et al (2002) The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci USA 4:16433–16437

    Article  Google Scholar 

  22. Pikaart M, Recillas-Targa F, Felsenfeld G (1998) Loss of transcriptional activity of the transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12:2852–2862

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Recillas-Targa F, Valadez-Graham V, Farrell CM (2004) Prospects and implications of using chromatin insulators in gene therapy and transgenesis. BioEssays 26:796–807

    Article  CAS  PubMed  Google Scholar 

  24. Furlan-Magaril M, Rebollar E, Guerrero G, Fernández A, Moltó E, González-Buendía E et al (2011) An insulator embedded in the Chicken α-globin locus regulates chromatin domain configuration and differential gene expression. Nucleic Acids Res 39:89–103

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Splinter E, Heath H, Kooren J, Palstra RJ, Klaus P, Grosveld F et al (2006) CTCF mediates long-range chromatin loop and local histone modification in the β-globin locus. Genes Dev 20:2349–2354

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao Z et al (2006) CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA 103:10684–10689

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. van Bortle K, Corces VG (2013) The role of chromatin insulators in nuclear architecture and genome function. Curr Opin Genet Dev 23(2):212–218

    Article  PubMed Central  PubMed  Google Scholar 

  28. Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W et al (2008) Domain organization of human chromosomes revealed by mapping the nuclear lamina interactions. Nature 453:948–951

    Article  CAS  PubMed  Google Scholar 

  29. Bonetta L (2005) The inside scoop-evaluating gene delivery methods. Nat Methods 2:875–883

    Article  CAS  Google Scholar 

  30. Sambrook J, Russell DW. Molecular cloning, a laboratory manual, 2(4):15.1–15.5

    Google Scholar 

  31. Hughes D, Marendy E, Dickerson C, Yetming K, Sample C, Sample J (2012) Contribution of CTCF and DNA methyltransferases DNMT1 and DNMT3B to Epstein-Barr Virus restricted latency. J Virol 86:1034–1045

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Ren L, Wang Y, Shi M, Wang X, Yang Z, Zhao Z (2012) CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation. PLoS One 7:e31416

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J et al (2006) Position-specific chemical modification increases specificity of siRNA-mediated gene silencing. RNA 12:1197–1205

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Chen PY, Weinmann L, Gaidatzis D, Pei Y, Zavolan M, Tuschl T et al (2008) Strand-specific 5′O-methylation of siRNA duplexes controls guide strand selection and target specificity. RNA 2:263–274

    Google Scholar 

  35. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y et al (2006) 3′-UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3:199–204

    Article  CAS  PubMed  Google Scholar 

  36. Anderson EM, Birmingham A, Baskerville S, Reynolds A, Maksimova E, Leake D et al (2008) Experimental validation of the importance of seed frequency to siRNA specificity. RNA 14:853–861

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Hou C, Zhao H, Tanimoto K, Dean A (2008) CTCF-dependent enhancer-blocking by alternative chromatin loop formation. Proc Natl Acad Sci USA 105:20398–20403

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Balakrishnan S, Witcher M, Berggren T, Emerson B (2012) Functional and molecular characterization of the role of CTCF in human embryonic stem cell biology. PloS One 7:e42424

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Klimatcheva E, Ronsenblatt JD, Planelles V (1999) Lentiviral vectors and gene therapy. Front Biosci 4:D481–96

    Article  CAS  PubMed  Google Scholar 

  40. Biasco L, Baricordi C, Aiuti A (2012) Retroviral integrations in gene therapy trials. Mol Ther 20:709–716

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Markusic D, Seppen J (2010) Doxycycline regulated lentiviral vectors. Methods Mol Biol 614:69–76

    Article  CAS  PubMed  Google Scholar 

  42. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 97:7963–7968

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333

    Article  CAS  PubMed  Google Scholar 

Further Reading

Download references

Acknowledgements

We acknowledge Ricardo Saldaña-Meyer for comments and critical reading of the manuscript. This work was supported by the Dirección General de Asuntos del Personal Académico-Universidad Nacional Autónoma de México (IN209403 and IN203811), Consejo Nacional de Ciencia y Tecnología, México (CONACyT; 42653-Q and 128464), and a Ph.D. fellowship from CONACyT and Dirección General de estudios de Posgrado-Universidad Nacional Autónoma de México (DGEP) (EG-B: 207989, RP-M: 173754, and EA-O: 256339). Additional support was provided by the Ph.D. Graduate Program, “Doctorado en Ciencias Biomédicas and Ciencias Bioquímicas.” We thank Fernando Suaste-Olmos for technical assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Félix Recillas-Targa .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

González-Buendía, E., Pérez-Molina, R., Ayala-Ortega, E., Guerrero, G., Recillas-Targa, F. (2014). Experimental Strategies to Manipulate the Cellular Levels of the Multifunctional Factor CTCF. In: Robles-Flores, M. (eds) Cancer Cell Signaling. Methods in Molecular Biology, vol 1165. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0856-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0856-1_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0855-4

  • Online ISBN: 978-1-4939-0856-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics