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Inhibition of NF-kB by ZAS3, a
zinc-finger protein that also
binds to the kB motif
Authors: Joung-Woo Hong, Carl E. Allen, and Lai-Chu Wu
Presenters:
Melissa Sherman
&
Troy Williams
Overview of NF-kB
• NF-kB pathway is used mainly during
inflammation and development
• ZAS and NF-kB are two distinct families of kBbinding proteins
• NF-kB is bound to inhibitory molecule IkB in the
cytoplasm
• Rel family of NF-kB encodes transcription
factors that regulate genes involved in:
– Immune responses
– Inflammation responses
– Antiapoptotic responses
Overview of NF-kB cont.
• Rel family of NF-kB differ from non-Rel-kBs by:
– Size
– Immunogenicity
– Sequence specificity to the kB motif
• Rel family interacts with the kB motif to induce gene
expression
• Rel family includes p65.p50 and p50.p50 dimers
NF-kB
p65
p50
kB motif = GGGACTTTCC
Basic NF-kB Pathway
• TNF is released
• TNF receptors on target cell bind
cytokine
• Trimerization of TNF occurs
• TRADD, RIP, and TRAF2 bind
to receptors
• IKKK is activated
• IKK is phosphorylated
• IkB is phophorylated
• IkB releases NF-kB
– IkB is ubiquitylated
– NF-kB transported to nucleus
• NF-kB binds to promoters (kB
motif) and activates transcription
Overview of ZAS
• ZAS proteins are large zinc-finger
transcriptional proteins used in:
– Growth
– Signal transduction
– Lymphoid development
• ZAS contains:
– Pair of zinc fingers
– Acidic region
– Serine/threonine-rich sequence
• A zinc finger is part of a protein that
can bind to DNA
Overview of ZAS cont.
•
•
•
•
There are three ZAS proteins: ZAS1, 2, and 3
ZAS3 is present in B lymphocytes
ZAS fusion proteins can bind to kB motif
ZAS3 inhibits NF-kB activation by:
– Inhibition of nuclear translocation of p65
– Competition for kB gene regulatory elements
– Repression of target gene transcription
Inhibited NF-kB Pathway
• TNF is released and binds to
TNF receptors
• Trimerization occurs
• TRADD, RIP, TRAF2, and
FADD bind to receptors
– FADD is an adaptor known to
transmit apoptotic and cell
proliferation signals
• ZAS3 binds to TRAF2
preventing IKK complex
– Nuclear translocation of p65
prevented
• ZAS3 may compete for kB
gene regulatory elements or
repress transcription
The Experiment
• Previous studies show that ZAS3 associates with
TRAF2 to inhibit NF-kB activation in cytoplasm
• Does ZAS 3 inhibit NF-kB activation in the nucleus?
• Observed that ZAS binds specifically to kB-like
sequences
• Are ZAS proteins non-Rel-kBs?
• Show that non-Rel-kB is absent in ZAS3-/- cells
• Show that NF-kB is expressed in ZAS3-/- cells
• Mechanism provides a checkpoint to control the
reprogramming of gene expression
EMSAs
• Electrophoretic Mobility-Shift Assays (EMSAs)
• B lymphocyte cell lines
• 32P-kB probe yielded several sequence-specific DNAprotein complexes (C1, C2, and C3)
EMSAs
• Addition of ZAS3 antiserum diminished C1
• p65 and p50 antibodies altered DNA-protein complex
patterns
–C2 likely p50.p50
–C3 likely p65.p50
•Unlabeled kB
oligonucleotides
diminished all
complexes
•Sp1 oligonucleotides
were noncompetitive
EMSAs
• ZAS3 knocked out by homologous recombination
(ZAS-/-B1 and ZAS3-/-B2)
• Prominent p65.p50 complex observed
• C1 was not observed
• Conclusion: ZAS most likely C1
EMSAs
• LPS is a reagent used to stimulate NF-kB in B lymphocytes
•ZAS3-/- cells: NF-kB DNA-binding was mostly found in
nuclear extracts
• LPS had minimal effect
•ZAS3+/+ cells: NF-kB
DNA-binding was found in
both nuclear and cytoplasmic
extracts
•LPS reduced cytoplasmic
binding and increased nuclear
binding
Immunoblot
• Nuclear p65 levels are 5
times higher in ZAS-/- cells
than ZAS+/+ cells
• LPS increased nuclear p65
levels significantly in
ZAS+/+ cells, but minimally
in ZAS -/- cells
• Conclusion: Supports results
of higher NF-kB DNAbinding activity in the
nucleus
Reporter Gene Assays
• kB-reporter activity was 30fold higher in ZAS3-/- than
ZAS+/+
• LPS increased kB-reporter
activity heavily in 38B9 cells
and minimally in ZAS-/- cells
• Due in part to the upregulation of nuclear p65
• Conclusion: ZAS3 has major
impact on NF-kB-dependent
gene expression and cell fate
Immunoblot
• Amount of TRAF1 and
TRAF2 was higher in
ZAS3-/- cells than 38B9
cells
• Conclusion: Expression
of TRAF1 and TRAF2
controlled by NF-kB
Immunoblot
• Amount of IkB-alpha in 38B9
cells was significantly higher
than that of ZAS3-/- cells
• Conclusion:
– In ZAS3-/- cells, the absence of
ZAS3 activates the assembly of
the IKK complex
– IKK phosphorylates IkB, IkB and
NF-kB separate, and IkB is
ubiquitylated
– In ZAS+/+ cells, IkB is never
degraded and shows a higher
concentration of IkB
Immunoblot
• A DNA fragment (with all
of the structural parts of
ZAS3) was inserted into a
FLAG-tag expression
plasmid.
• It yielded abundant fusion
proteins.
• Conclusion: ZAS3 was
also found more in the
nucleus then the cytoplasm.
Reporter Gene Assays
• Fig. 3B- Reporter gene assays initially
showed that ZAS3 expression reduced
the activity of kB- reporter genes in HEK
293
• Fig. 3C- Expression constructs showed
that ZAS3 repressed NF-kB-mediated
transactivation of the kB-reporter.
• Fig. 3D- ZAS3 expression also inhibited
the kB reporter in ZAS3 -/- cells.
• Conclusion: They all provide a link
between ZAS3 deficiency and NF-kB
activation.
Immunoblot
•
In 38B9 cells, p65 was mainly located
in the cytoplasmic extracts and barely
in the nuclear ones
•
In ZAS3, p65 was mainly found in the
nuclear extracts.
•
Introduction of ZAS3 expression
construct caused an increase of
expression in the cytoplasm and less
activity in the nucleus.
•
H1 (a nuclear protein) was a control
for the experiment
•
Conclusion: p65 was regulated in
ZAS3 -/- cells, and that ZAS3
activated nuclear export pathway of
p65
kB-Reporter Gene Assays
• kB-reporter was much higher in
p65+/+ cells
– Demonstrates importance of p65 in
kB-mediated transactiviation
• When p65 was absent, kB-reporter
gene activity was still higher than
control
– kB-reporter must be driven by
transacting factors in kB-DNA
• Conclusion: ZAS3 represses kBmediated transcription
independent of p65
kB-Reporter Gene Assays
• Expression vectors were
cotransfected with reporter plasmid
pCMV110, upstream CAT gene
• Expression of the CAT reporter was
increased 50-fold by plasmid pSGVP16
– VP16 domain is a strong transcriptional
activator
• Expression of the CAT reporter was
repressed by plasmid pSG-ZASC
• Conclusion: ZAS3 inhibits
transactivation of VP16 and so may
function as a transcriptional
repressor
Conclusions
• Previous studies have determined ZAS helps to maintain
normal growth. Expression of ZAS restricts proliferation
and mitosis of germ cells
• Down-regulation of ZAS is associated with accelerated
growth of cell lines and growth of multinucleated giant cells
• ZAS3 required maturation (prevents apoptosis)
• In ZAS-/- cells, developmental abnormalities resulted
including tumorigenesis, polydactyly, and hydronephrosis
• Changes of ZAS gene expression have been associated with
leukemia patients
– Suggest ZAS3 contains tumor suppression function
• ZAS proteins have been implicated in signal transduction
• ZAS3 associates with TRAF2 to repress TNF/NF-kB
pathway
Conclusions cont.
• Localization of p65 and transactivation activity of NFkB are modulated by ZAS3
• Where NF-kB activates transcription, ZAS3 mostly
represses transcription
• ZAS3 is responsible partly for guarding NF-kBmediated transactivation by:
– Inhibiting nuclear localization of p65
– Competing for kB gene regulatory elements
– Serving as a transcriptional repressor
Conclusions cont.
• ZAS family of zinc-finger proteins are
governors of NF-kB-mediated cell functions
including:
–
–
–
–
Apoptosis
Proliferation
Inflammation
Immunity
• Non-Rel-kB most likely related to ZAS3