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

6000

500

5000

500

6000

5000

6000

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.