Biomolecules at Kenyon XX HHMI at Kenyon xx Jmol Home xx Biology Dept xx COMMENTS and CORRECTIONS

Zif268 Zinc Finger Transcription Factor

Robert Carpenter '10 and Emily Nutt '10


I. Introduction to Zif268

Zif268 is a transcription factor coded by an immediate early gene. This transcription factor acts with other proteins to actively enhance or repress a gene by binding to the DNA at enhancer or repressor sites. Zif268 is one of the most easily expressed proteins because of its numerous vital functions that target many important genes. Zif268 regulates neurological genes and cellular growth and proliferation genes. The transcription factor is expressed in mammalian neurons during visual and fear learning, as well as in song learning in birds. Zif268 also responds to cellular growth and proliferation signals by enhancing the expression of TGFβ1, a protein which regulates cell differentiation and proliferation.

Zif268 binds to neuron and growth genes through a tandemly repeated zinc finger motif. All proteins with a zinc finger motif contain a consensus or linker sequence of Thr-gly-glu-lys. This sequence is seen between each of the three zinc fingers in Zif268. However the consensus sequence between fingers I and II is slightly different because it has a Gln in place of a Glu.


II. General Structure

Zif268 is a transcription factor with 88 amino acid residues. These amino acid residues form three zinc finger motifs . The Zif268 fingers interact with the DNA recognition sequence 5’GCG-TGG-GCG 3’. Finger I binds to the 3’ end of the recognition sequence and finger III binds to the 5’ end of the recognition sequence . Each finger contains two antiparallel beta sheets and an a-helix, both of which interact with a zinc atom . The two β-sheets in each finger have different functionality. The first β-sheet has no interaction with DNA, while the second β-sheet interacts with the B-DNA backbone. All three fingers can be superimposed on one another because they have identical secondary structure.

A zinc finger is stabilized by zinc interactions and hydrophobic interactions. In each finger, two cysteine residues on the beta sheet and two histidine residues on the α-helix interact with the zinc ion .  Highly conserved hydrophobic residues from the a-helix and beta-sheets help to stabilize the zinc finger motif . PHE 16, LEU 22, and HIS 25, stabilize the zinc finger by forming a hydrophobic pocket which shields the zinc ion from water .   The hydrophobic interactions in finger I occur between threonine's oxygen and a nitrogen of the peptide backbone . Other conserved hyrophobic residues form a hydrophobic pocket on both sides of the finger to sheild the zinc ion from water . Hydrogen bonding also helps to stabilize the secondary structure so that the protein residues can interact with the zinc ion. The stability created by both zinc ion interactions and hydrogen bonding within the secondary structure allows the protein residues to interact with the DNA bases and backbone. Hydrogen binding to the amine group of the protein backbone by ILE , THR and GLU , stabilizes the secondary structure. These hydrophobic and zinc interactions help stabilize the protein so that it can effectively bind to DNA.


III. DNA Interactions

In Zif268, the α-helix and a beta-sheet of each finger interact with B-DNA. The α-helix primarily interacts with B-DNA bases, and the β-sheet interacts with the B-DNA backbone.

Zif268 makes 11 different hydrogen bond interactions with B-DNA bases. All three fingers directly interact with the guanine rich strand of the recognition sequence. They also interact with the cytosine rich strand through water mediated interactions. Finger I and III each make four critical hydrogen bonds with B-DNA, while finger II only makes three. All three fingers have an arginine immediatly proceeding the α-helix, this residue binds the N7 and O6 of guanine . Fingers I and III both have an additional arginine - guanine interactions at the fourth residue of the a-helix . The second residue of the α-helix in all three fingers is an aspartic acid residue, which acts to stabilize the arginine - guanine interaction. The aspartic acid stabilizes the interaction by hydrogen bonding to two nitrogens on the end of the arginine residue .

The a-helix of finger II binds to DNA with slightly different amino acids from those of fingers I and III. As in fingers I and III, the arginine immediately preceding the a-helix in finger II binds to guanine and is stabilized by an aspartic acid from the a-helix . The third residue of the a-helix is a histidine that binds to DNA and enhances specificity. This histidine interculates between guanine and thymine, forms a hydrogen bond with N7 with the guanine , and has van der Waals interactions with thymine . The stacking ability of histidine allows Zif268 to bind specifically to the recognition sequence.

Not only do the α-helix residues interact with the guanine rich strand of the recognition helix, but they also make water mediated interactions with the cytosine rich strand. The second residue on each α-helix, aspartic acid , not only hydrogen bonds with arginine, but interacts with guanine's complementary base pair cytosine through a water mediated interaction . A water mediated interaction also occurs between the arginine that lies immediately before the alpha helix, and the guanine backbone .

Residues on both the α-helix and a beta-sheet of each finger hydrogen bond with the phosphate backbone. The histidine on each α-helix that interacts with the zinc ion also hydrogen bonds with the phosphate backbone. This histidine uses a water mediated interaction to contact an oxygen in the phosphate group. An arginine on the second β-sheet of each finger also hydrogen bonds with the phosphate backbone . These protein-DNA backbone interactions allow for Zif268 to bind more specifically and strongly to the recognition sequence.

IV. Zif268 in Neurons

Zif268 is involved in mammalian neuronal plasticity and therefore learning. Zif268 is consitutivly expressed in several parts of the mammalian brain, one of which is the temporal lobe. A mammalian neuron must be changed in order for learning to occur, and Zif268 plays a role in the alteration of neuron. For example the increase of calcium ions in a neuron can turn on Zif268. Although scientists do not know exactly how Zif268 contributes to memory, they do know that it is present during several types of memory and learning, visual learning, and fear learning. Several experiments show that Zif268 is expressed when visual and fearful memories are created. When mammals learn something visually, Zif268 is highly expressed in the temporal lobe, the part of the brain involved with memory. If Zif268 is up-regulated in the temporal lobe during the creation of memories, it can be assumed that it plays a role in memory creation. Zif268 is not involved in memory recollection, as it is not expressed when the same visual memory is recalled. Zif268 is also highly expressed when mammals create a memory due to fear. The expression of Zif268 during creation of memories leads scientists to believe that it must effect neurons in such a way that a long term memory is created.

Many scientist believe that in order for a long term memory to be stored it must go through a process where it is activated again and further stored (Bozon et al 2003). Bozon et al (2003) demonstrated the importance of Zif268 in formation of long term memories. Wild-type mice and mice with a mutant Zif268 gene were exposed to a box with three objects. After 24 hours one of the objects was changed, and both types of mice remembered the old objects and therefore spent time investigating the new object. However, if the scientists waited longer than 24 hours, the mutant mice did not remember the old objects and spent the same amount of time investigating each object. These tests showed that Zif268 is needed to retain a memory for a long period of time. Exposing the mutant mice to the objects multiple times before the object was changed helped the mutant mice to retain the memory. This demonstrated that Zif268 mutant mice are able to retain a long term memory if exposed to that memory multiple times.


V. Effect on Cancer

Zif268 has important cancer prevention functionality because it decreases cancer cell growth, and the presence of the Zif268 gene prevents the onset of acute myeloid leukemia. Zif268 prevents cell growth by activating TGFβ1, which is a protein that regulates cell proliferation and differentiation. Studies by Lui et al (1996) showed that an increase of TGFβ1 in cancer cells greatly decreased cellular growth. They also found that when more Zif268 was present in the cell more TGFβ1 was produced. The greater production of TGFβ1 prevented cell proliferation, suggesting that Zif268 is a protein that could be used for cancer prevention.

Zif268's role in cancer prevention can also be seen when there is a mutation to its gene on chromosome V. When a chromosome mutation occurs and a person does not have the gene for Zif268, they experience the onset of acute myeloid leukemia or myelodysplastic syndrome. If chromosome V is mostly or completely missing from a genome, the individual will first contract myelodysplastic syndrome which then leads to acute myeloid leukemia. Myelodysplasic syndrome is a series of disorders that all affect the bone marrow. This disease starts from a single affected bone marrow stem cell, which in turn produces a series of abnormal myeloid cells: red blood cells, white blood cells and platelets. Myelodysplastic syndrome affects mainly red blood cells, but this syndrome can eventually lead to acute myeloid leukemia or cancer of the white blood cells. A loss of Zif268 leads to abnormal myeloid cells and ultimately to myelodysplastic syndrome and even acute myeloid leukemia.

VI. References

Davis, Sabrina, Bozon, Bruno, Laroche, Serge. 2002. How Necessary is the Activation of the Immediate Early Gene Zif268 in synaptic plasticity and learning? Behavioral Brain Research. 142: 17-30.

Elrod-Erickson, Monica, Rould, Mark A., Nekludova, Lena, Pabo, Carl O. 1996. Zif268 protein-DNA complex refined at 1.6 Å: a model system for understanding zinc finger-DNA interactions. Elsevier Science. 3: 1171-1180.

Knapska, Ewwlina and Kacmarek, Leszek. 2004. A Gene for Neuronal Plasticity in the Mammalian Brain: Zif268/ Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Progress in Neurobiology. 74: 183-211.

Liu, C, Adamson, E, Mercola, D. 1996. Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta-1. Proc. Nat. Acad. Sci. 93: 11831-11836.

Pavletich, Nikola P. and Pabo, Carl O. 1991. Zinc finger-DNA Recognition: Crystal Structure of a Zif268-DNA Complex at 2.1 Å. Science 252: 809-817.

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