Transcript Zinc Finger

ATF
versity of Wisconsin-Madison Team Members
Nathan Klapoetke
Sean McMaster
David Peterson
Our Vision
Interface Criteria
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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
Zinc finger
2) Effector domain (ED), recruits
cellular machinery to site where
DBD has bound
ED
DBD
++
or
--
Activate or
repress gene
transcription
Zinc Finger
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Made of 30 amino acids
Chelated by 1 zinc ion
Protein structure (beta-beta-alpha)
Zinc Finger
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Recognizes 3 base pairs of DNA
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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.
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Greatly increases DNA binding
specificity
*5
ATFα
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Obtained from Carlos
Barbas III
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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
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Reformatting of ATFα
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Cloning
Site-directed mutagenesis
Analysis
Design is specific to the Bcl-2 promoter
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Exploited in subsequent experiments
Cloning
6000
6000
5000
5000
500
500
Digestion of Clones
6000
5000
Mutagenesis
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Changing amino acids to conform to ideal
binding sites (*1, *2)
Existing ZiF Design Tools
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Based on meta-analysis of the “GNN” family
Zif (*1, *2)
ATFΩ
Limitations of ZiF Tools
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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
*3
•Unbiased
•Probe entire N-mer sequence space
9-mer Array
1nM
1:1000
1x
50uM
0.01%
ZiF
HA antibody
HOX
Zinc Acetate
Triton X
CW115-IG
9mer array
Binding Affinity
Position Weight Matrix
*4
ATF test system
Why regulate Bcl-2?
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“… 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
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“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
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“… balancing Bcl-2 to Bax in
Bcl-2 Regulation
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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
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Probing for presence of ATF:
ATF
+TET
ATF
vector
(-) ctrl
} ~23kd
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Probing for change in Bcl-2 protein levels:
vector
vector
+TET
ATF
ATF
+TET
} ~26kd
Future in vivo testing
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Work in bacteria,
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Mammalian cells are complicated
Target a different pathway
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Bcl-2 family of proteins are complicated
Conclusion
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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
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1. GNN Dreier B, Segal DJ, Barbas CF 3rd. Insights into the molecular recognition of the 5'-GNN3' 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. 14:11881190.
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.