Download - ATF
University of Wisconsin-Madison Team MembersNathan Klapoetke
Sean McMasterDavid Peterson
ATF
Our Vision
Interface Criteria
Modular design Compatible with bacterial and
mammalian systems Well characterized Reliable
What is ATF?
1) DNA binding domain (DBD), recognizes specific DNA sequence and binds to it
2) Effector domain (ED), recruits cellular machinery to site where DBD has bound
DBD
ED
++ or - -
Zinc finger
Activate or repress gene transcription
Zinc Finger
Made of 30 amino acids Chelated by 1 zinc ion Protein structure (beta-beta-alpha)
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
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
ATFΩ
Reformatting of ATFα Cloning Site-directed mutagenesis Analysis
Design is specific to the Bcl-2 promoter Exploited in subsequent experiments
Cloning
5000
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Digestion of Clones
Mutagenesis
Changing amino acids to conform to ideal binding sites (*1, *2)
Existing ZiF Design Tools
Based on meta-analysis of the “GNN” family Zif (*1, *2)
ATFΩ
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
•Unbiased
•Probe entire N-mer sequence space
*3
9-mer Array1nM ZiF1:1000 HA antibody1x HOX50uM Zinc Acetate0.01% Triton X
CW115-IG9mer array
Binding Affinity
Position Weight Matrix
*4
ATF test system
Why regulate Bcl-2?
“… manipulation of the Bcl-2 system may also provide new treatments to forestall 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
Oxidative stress (such as caused by Doxorubicin) induces apoptosis
ATFΩ is designed to inhibit apoptosis
ATF in action
*Assay for function in mouse cells
Western Blot Results
Probing for presence of ATF:
Probing for change in Bcl-2 protein levels: vector vector ATF ATF +TET +TET
ATF ATF vector (-) ctrl+TET
} ~23kd
} ~26kd
Future in vivo testing
Work in bacteria, Mammalian cells are complicated
Target a different pathway Bcl-2 family of proteins are complicated
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 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
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.