Zif268
Zinc Finger Transcription Factor
Robert Carpenter '10 and Emily Nutt '10
Contents:
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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