1. 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

2. 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

3. 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 p65p50 NF-kB

4. 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

5. 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

6. 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

7. 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

8. 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

9. EMSAs Electrophoretic Mobility-Shift Assays (EMSAs) B lymphocyte cell lines 32 P-kB probe yielded several sequence-specific DNA- protein complexes (C1, C2, and C3)

10. 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

11. 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

12. 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

13. 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

14. 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

15. Immunoblot Amount of TRAF1 and TRAF2 was higher in ZAS3-/- cells than 38B9 cells Conclusion: Expression of TRAF1 and TRAF2 controlled by NF-kB

16. 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

17. 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.

18. 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.

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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