Genome-wide siRNA screen for mediators of NF-κB activation

Benjamin E. Gewurz, Fadi Towfic, Jessica C. Mar, Nicholas P. Shinners, Kaoru Takasaki, Bo Zhao, Ellen D. Cahir-McFarland, John Quackenbush, Ramnik J. Xavier, Elliott Kieff

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.

Original languageEnglish (US)
Pages (from-to)2467-2472
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number7
DOIs
StatePublished - Feb 14 2012
Externally publishedYes

Fingerprint

Small Interfering RNA
Genome
Interleukin-1
Proteins
Phosphotransferases
HEK293 Cells
Human Genome
Enzymes
Ligases
Ubiquitin
RNA Interference
Human Herpesvirus 4
Phosphoric Monoester Hydrolases
Cell Differentiation
Cell Survival
Membrane Proteins
Cell Proliferation
Messenger RNA

Keywords

  • Cytokine
  • Inflammation
  • Lymphoma
  • Oncogene
  • Signaling

ASJC Scopus subject areas

  • General

Cite this

Gewurz, B. E., Towfic, F., Mar, J. C., Shinners, N. P., Takasaki, K., Zhao, B., ... Kieff, E. (2012). Genome-wide siRNA screen for mediators of NF-κB activation. Proceedings of the National Academy of Sciences of the United States of America, 109(7), 2467-2472. https://doi.org/10.1073/pnas.1120542109

Genome-wide siRNA screen for mediators of NF-κB activation. / Gewurz, Benjamin E.; Towfic, Fadi; Mar, Jessica C.; Shinners, Nicholas P.; Takasaki, Kaoru; Zhao, Bo; Cahir-McFarland, Ellen D.; Quackenbush, John; Xavier, Ramnik J.; Kieff, Elliott.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 7, 14.02.2012, p. 2467-2472.

Research output: Contribution to journalArticle

Gewurz, BE, Towfic, F, Mar, JC, Shinners, NP, Takasaki, K, Zhao, B, Cahir-McFarland, ED, Quackenbush, J, Xavier, RJ & Kieff, E 2012, 'Genome-wide siRNA screen for mediators of NF-κB activation', Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 7, pp. 2467-2472. https://doi.org/10.1073/pnas.1120542109
Gewurz, Benjamin E. ; Towfic, Fadi ; Mar, Jessica C. ; Shinners, Nicholas P. ; Takasaki, Kaoru ; Zhao, Bo ; Cahir-McFarland, Ellen D. ; Quackenbush, John ; Xavier, Ramnik J. ; Kieff, Elliott. / Genome-wide siRNA screen for mediators of NF-κB activation. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 7. pp. 2467-2472.
@article{1ae784ca89284c09a1efc750d4310d6c,
title = "Genome-wide siRNA screen for mediators of NF-κB activation",
abstract = "Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.",
keywords = "Cytokine, Inflammation, Lymphoma, Oncogene, Signaling",
author = "Gewurz, {Benjamin E.} and Fadi Towfic and Mar, {Jessica C.} and Shinners, {Nicholas P.} and Kaoru Takasaki and Bo Zhao and Cahir-McFarland, {Ellen D.} and John Quackenbush and Xavier, {Ramnik J.} and Elliott Kieff",
year = "2012",
month = "2",
day = "14",
doi = "10.1073/pnas.1120542109",
language = "English (US)",
volume = "109",
pages = "2467--2472",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "7",

}

TY - JOUR

T1 - Genome-wide siRNA screen for mediators of NF-κB activation

AU - Gewurz, Benjamin E.

AU - Towfic, Fadi

AU - Mar, Jessica C.

AU - Shinners, Nicholas P.

AU - Takasaki, Kaoru

AU - Zhao, Bo

AU - Cahir-McFarland, Ellen D.

AU - Quackenbush, John

AU - Xavier, Ramnik J.

AU - Kieff, Elliott

PY - 2012/2/14

Y1 - 2012/2/14

N2 - Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.

AB - Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β-, and 79 for TNFα-mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β-, or TNFα-mediated canonical NFκB activation.

KW - Cytokine

KW - Inflammation

KW - Lymphoma

KW - Oncogene

KW - Signaling

UR - http://www.scopus.com/inward/record.url?scp=84857126437&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84857126437&partnerID=8YFLogxK

U2 - 10.1073/pnas.1120542109

DO - 10.1073/pnas.1120542109

M3 - Article

C2 - 22308454

AN - SCOPUS:84857126437

VL - 109

SP - 2467

EP - 2472

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 7

ER -