Inhibition of NF-kB by ZAS3, a zinc-finger protein that also

binds to the kB motif

Presenters:

Melissa Sherman

&

Troy Williams

Authors: Joung-Woo Hong, Carl E. Allen, and Lai-Chu Wu

Overview of NF-kB• NF-kB pathway is used mainly during

inflammation and development• ZAS and NF-kB are two distinct families of kB-

binding 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

kB motif = GGGACTTTCC

p65 p50

NF-kB

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 DNA-

protein 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 DNA-binding activity in the nucleus

Reporter Gene Assays• kB-reporter activity was 30-

fold higher in ZAS3-/- than ZAS+/+

• LPS increased kB-reporter activity heavily in 38B9 cells and minimally in ZAS-/- cells

• Due in part to the up-regulation 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 kB-mediated 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 pSG-VP16– 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 NF-

kB are modulated by ZAS3• Where NF-kB activates transcription, ZAS3 mostly

represses transcription• ZAS3 is responsible partly for guarding NF-kB-

mediated 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