1 / 28

ATF

ATF. University of Wisconsin-Madison Team Members Nathan Klapoetke Sean McMaster David Peterson. Our Vision. Interface Criteria. Modular design Compatible with bacterial and mammalian systems Well characterized Reliable. What is ATF?. DNA binding domain (DBD),

denis
Download Presentation

ATF

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. ATF University of Wisconsin-Madison Team Members Nathan Klapoetke Sean McMaster David Peterson

  2. Our Vision

  3. Interface Criteria • Modular design • Compatible with bacterial and mammalian systems • Well characterized • Reliable

  4. What is ATF? • DNA binding domain (DBD), recognizes specific DNA sequence and binds to it • Effector domain (ED), recruits cellular machinery to site where DBD has bound Zinc finger ++ or - - ED • Activate or repress gene transcription DBD

  5. Zinc Finger • Made of 30 amino acids • Chelated by 1 zinc ion • Protein structure (beta-beta-alpha)

  6. Zinc Finger • Recognizes 3 base pairs of DNA • Alpha-helix interacts with the DNA triplet • Some fingers can recognize 4 base pairs • Can covalently link fingers to recognize and bind to longer DNA sequence. • Greatly increases DNA binding specificity *5

  7. ATFα • Obtained from Carlos Barbas III • Binds to GGA-GTT-G** • 3 zinc fingers are linked by TGEKP • Has transcription factor VP64, a strong activator Original Zif extracted from pMX plasmid

  8. ATFΩ • Reformatting of ATFα • Cloning • Site-directed mutagenesis • Analysis • Design is specific to the Bcl-2 promoter • Exploited in subsequent experiments

  9. Cloning 6000 6000 5000 6000 5000 5000 500 500 Digestion of Clones

  10. Mutagenesis • Changing amino acids to conform to ideal binding sites (*1, *2)

  11. Existing ZiF Design Tools • Based on meta-analysis of the “GNN” family Zif (*1, *2)

  12. ATFΩ

  13. Limitations of ZiF Tools • Cannot accurately predict binding effects of covalently linked zinc fingers • Does not give binding affinity • Cannot screen for additional undesired binding sequences

  14. Cognate Site Identity (CSI) Array *3 • Unbiased • Probe entire N-mer sequence space

  15. 9-mer Array 1nM ZiF 1:1000 HA antibody 1x HOX 50uM Zinc Acetate 0.01% Triton X CW115-IG 9mer array

  16. Binding Affinity

  17. Position Weight Matrix *4

  18. ATF test system

  19. Why regulate Bcl-2? • “… manipulation of the Bcl-2 system may also provide new treatments toforestall cardio-myocyte apoptosis. The potential strategies could involve up-regulating the antiapoptotic Bcl-2 …” -Gill, Mestril, and Samali, 2002 • “Antiapoptotic Bcl-2 proteins have therapeutic potential for heart disease, since they have been shown to protect myocardial cells from various stresses.” -Gustafsson and Gottlieb, 2007 • “… balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.” -Tung et al., 2003

  20. Bcl-2 Regulation • Oxidative stress (such as caused by Doxorubicin) induces apoptosis • ATFΩ is designed to inhibit apoptosis

  21. ATF in action *Assay for function in mouse cells

  22. Western Blot Results • Probing for presence of ATF: • Probing for change in Bcl-2 protein levels: ATF ATF vector (-) ctrl +TET } ~23kd vector vector ATF ATF +TET +TET } ~26kd

  23. Future in vivo testing • Work in bacteria, • Mammalian cells are complicated • Target a different pathway • Bcl-2 family of proteins are complicated

  24. Conclusion • So, since we have begun, questions may have been created more questions than they have been answered. But, the ideas and possibilities that come from harnessing ATF technology was well worth it.

  25. References • 1. GNN Dreier B, Segal DJ, Barbas CF 3rd. Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains. J Mol Biol. 2000 Nov 3;303(4):489-502. • 2. GNN Segal DJ, Dreier B, Beerli RR, Barbas CF 3rd. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758-63. • 3. C. L. Warren et al. 2005. Defining the sequence-recognition profile of DNA-binding molecules. Proc. Natl. Acad. Sci. USA. 103: 867-872. • 4. Crooks, Gavin et al. 2004. WebLogo: A Sequence Logo Generator. Genome Res. 14:1188-1190. • 5. Luscombe, Nicholas, et al (9 June 2000). "An overview of the structures of protein-DNA complexes." Genome Biology Review 1 (1): 4-5. • 6. Serebriiskii et al., 2007 • 7. Adams and Cory, 2001 • 8. Torsten Wittman, UCSF

  26. Thanks Advisors: Aseem Ansari, Franco Cerrina, & Doug Weibel We would like to thank the following people for their suggestions and aid in the implementation of this project: Clayton Carlson, Leslie Donato, Chris Warren, Mary Ozers We would like to thank the College of Engineering, Nanoscale Science and Engineering Center, and Chancellor John Wiley for their financial support.

More Related