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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),

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Presentation Transcript
slide2

ATF

University of Wisconsin-Madison Team Members

Nathan Klapoetke

Sean McMaster

David Peterson

interface criteria
Interface Criteria
  • Modular design
  • Compatible with bacterial and mammalian systems
  • Well characterized
  • Reliable
what is atf
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

zinc finger
Zinc Finger
  • Made of 30 amino acids
  • Chelated by 1 zinc ion
  • Protein structure (beta-beta-alpha)
zinc finger1
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

slide8
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

slide9
ATFΩ
  • Reformatting of ATFα
    • Cloning
    • Site-directed mutagenesis
    • Analysis
  • Design is specific to the Bcl-2 promoter
    • Exploited in subsequent experiments
cloning
Cloning

6000

6000

5000

6000

5000

5000

500

500

Digestion of Clones

mutagenesis
Mutagenesis
  • Changing amino acids to conform to ideal binding sites (*1, *2)
existing zif design tools
Existing ZiF Design Tools
  • Based on meta-analysis of the “GNN” family Zif (*1, *2)
limitations of zif tools
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
cognate site identity csi array
Cognate Site Identity (CSI) Array

*3

  • Unbiased
  • Probe entire N-mer sequence space
9 mer array
9-mer Array

1nM ZiF

1:1000 HA antibody

1x HOX

50uM Zinc Acetate

0.01% Triton X

CW115-IG

9mer array

why regulate bcl 2
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

bcl 2 regulation
Bcl-2 Regulation
  • Oxidative stress (such as caused by Doxorubicin) induces apoptosis
  • ATFΩ is designed to inhibit apoptosis
atf in action
ATF in action

*Assay for function in mouse cells

western blot results
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

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

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