Publications by authors named "Brian Zarnegar"

27 Publications

  • Page 1 of 1

Structural modularity of the XIST ribonucleoprotein complex.

Nat Commun 2020 12 2;11(1):6163. Epub 2020 Dec 2.

Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, 94305, USA.

Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and mA modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and mA modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies.
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http://dx.doi.org/10.1038/s41467-020-20040-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710737PMC
December 2020

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation.

Elife 2020 05 7;9. Epub 2020 May 7.

Center for Personal Dynamic Regulomes, Stanford University, Stanford, United States.

The lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an -binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and RNA transcription. Spen binds directly to RNAs that show structural similarity to the A-repeat of , a region critical for -mediated gene silencing. RNA and A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of RNA to Spen and results in strictly local gene silencing in . These results suggest that may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.
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http://dx.doi.org/10.7554/eLife.54508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282817PMC
May 2020

Genetic and genomic studies of pathogenic EXOSC2 mutations in the newly described disease SHRF implicate the autophagy pathway in disease pathogenesis.

Hum Mol Genet 2020 03;29(4):541-553

Department travellers of Pathology, Stanford School of Medicine, Stanford, CA 94305, USA.

Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.
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http://dx.doi.org/10.1093/hmg/ddz251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068030PMC
March 2020

Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.

Cell 2019 01 20;176(1-2):361-376.e17. Epub 2018 Dec 20.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA. Electronic address:

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
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http://dx.doi.org/10.1016/j.cell.2018.11.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329648PMC
January 2019

RNA-protein interaction detection in living cells.

Nat Methods 2018 03 5;15(3):207-212. Epub 2018 Feb 5.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.
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http://dx.doi.org/10.1038/nmeth.4601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886736PMC
March 2018

CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue.

Dev Cell 2017 10 21;43(2):227-239.e5. Epub 2017 Sep 21.

Program in Epithelial Biology, Stanford University, 269 Campus Drive, Room 2145, Stanford, CA 94305, USA; Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA. Electronic address:

Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.
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http://dx.doi.org/10.1016/j.devcel.2017.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659279PMC
October 2017

irCLIP platform for efficient characterization of protein-RNA interactions.

Nat Methods 2016 06 25;13(6):489-92. Epub 2016 Apr 25.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.
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http://dx.doi.org/10.1038/nmeth.3840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477425PMC
June 2016

Network Analysis Identifies Mitochondrial Regulation of Epidermal Differentiation by MPZL3 and FDXR.

Dev Cell 2015 Nov;35(4):444-57

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA. Electronic address:

Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.
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http://dx.doi.org/10.1016/j.devcel.2015.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845910PMC
November 2015

The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer.

Nat Genet 2016 Jan 23;48(1):53-8. Epub 2015 Nov 23.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.
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http://dx.doi.org/10.1038/ng.3452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324971PMC
January 2016

A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation.

Dev Cell 2015 Mar;32(6):693-706

Program in Epithelial Biology, Stanford University, Stanford, CA 94305, USA; Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA. Electronic address:

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.
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http://dx.doi.org/10.1016/j.devcel.2015.01.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456036PMC
March 2015

Dissecting noncoding and pathogen RNA-protein interactomes.

RNA 2015 Jan 19;21(1):135-43. Epub 2014 Nov 19.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.
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http://dx.doi.org/10.1261/rna.047803.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274633PMC
January 2015

IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.

Nat Med 2013 May 21;19(5):626-630. Epub 2013 Apr 21.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA.

Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif-containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.
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http://dx.doi.org/10.1038/nm.3165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190012PMC
May 2013

Genetic pathways in disorders of epidermal differentiation.

Trends Genet 2013 Jan 8;29(1):31-40. Epub 2012 Nov 8.

Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA.

More than 100 human genetic skin diseases, impacting over 20% of the population, are characterized by disrupted epidermal differentiation. A significant proportion of the 90 genes identified in these disorders to date are concentrated within several functional pathways, suggesting the emergence of organizing themes in epidermal differentiation. Among these are the Notch, transforming growth factor β (TGFβ), IκB kinase (IKK), Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), p63, and Wnt signaling pathways, as well as core biological processes mediating calcium homeostasis, tissue integrity, cornification, and lipid biogenesis. Here, we review recent results supporting the central role of these pathways in epidermal differentiation, highlighting the integration of genetic information with functional studies to illuminate the biological actions of these pathways in humans as well as to guide development of future therapeutics to correct their dysfunction.
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http://dx.doi.org/10.1016/j.tig.2012.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477429PMC
January 2013

Genomic profiling of a human organotypic model of AEC syndrome reveals ZNF750 as an essential downstream target of mutant TP63.

Am J Hum Genet 2012 Sep 23;91(3):435-43. Epub 2012 Aug 23.

Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.
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http://dx.doi.org/10.1016/j.ajhg.2012.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511987PMC
September 2012

ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation.

Dev Cell 2012 Mar 23;22(3):669-77. Epub 2012 Feb 23.

Department of Medicine/Cellular and Molecular Medicine, University of California-San Diego School of Medicine, La Jolla, CA 92093, USA.

Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.
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http://dx.doi.org/10.1016/j.devcel.2011.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306457PMC
March 2012

Negative feedback in noncanonical NF-kappaB signaling modulates NIK stability through IKKalpha-mediated phosphorylation.

Sci Signal 2010 May 25;3(123):ra41. Epub 2010 May 25.

Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, 609 Charles E. Young Drive East, Los Angeles, CA 90095, USA.

Canonical and noncanonical nuclear factor kappaB (NF-kappaB) signaling are the two basic pathways responsible for the release of NF-kappaB dimers from their inhibitors. Enhanced NF-kappaB signaling leads to inflammatory and proliferative diseases; thus, inhibitory pathways that limit its activity are critical. Whereas multiple negative feedback mechanisms control canonical NF-kappaB signaling, none has been identified for the noncanonical pathway. Here, we describe a mechanism of negative feedback control of noncanonical NF-kappaB signaling that attenuated the stabilization of NF-kappaB-inducing kinase (NIK), the central regulatory kinase of the noncanonical pathway, induced by B cell-activating factor receptor (BAFF-R) and lymphotoxin beta receptor (LTbetaR). Inhibitor of kappaB (IkappaB) kinase alpha (IKKalpha) was previously thought to lie downstream of NIK in the noncanonical NF-kappaB pathway; we showed that phosphorylation of NIK by IKKalpha destabilized NIK. In the absence of IKKalpha-mediated negative feedback, the abundance of NIK increased after receptor ligation. A form of NIK with mutations in the IKKalpha-targeted serine residues was more stable than wild-type NIK and resulted in increased noncanonical NF-kappaB signaling. Thus, in addition to the regulation of the basal abundance of NIK in unstimulated cells by a complex containing tumor necrosis factor receptor-associated factor (TRAF) and cellular inhibitor of apoptosis (cIAP) proteins, IKKalpha-dependent destabilization of NIK prevents the uncontrolled activity of the noncanonical NF-kappaB pathway after receptor ligation.
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http://dx.doi.org/10.1126/scisignal.2000778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913610PMC
May 2010

Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK.

Nat Immunol 2008 Dec 9;9(12):1371-8. Epub 2008 Nov 9.

Department of Microbiology, Immunology & Molecular Genetics, University of California Los Angeles, 609 Charles E. Young Drive East, Los Angeles, California 90095, USA.

Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.
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http://dx.doi.org/10.1038/ni.1676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2676931PMC
December 2008

Control of canonical NF-kappaB activation through the NIK-IKK complex pathway.

Proc Natl Acad Sci U S A 2008 Mar 21;105(9):3503-8. Epub 2008 Feb 21.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.

Articles in recent years have described two separate and distinct NF-kappaB activation pathways that result in the differential activation of p50- or p52-containing NF-kappaB complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-kappaB activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-kappaB pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-kappaB activation and gene expression in vitro and in vivo. We also demonstrate that deregulation of the canonical NF-kappaB pathway in TRAF3-deficient cells results from accumulation of NF-kappaB-inducing kinase (NIK), the essential kinase mediating noncanonical NF-kappaB activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-kappaB activation pathways.
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http://dx.doi.org/10.1073/pnas.0707959105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265190PMC
March 2008

IRF3-dependent type I interferon response in B cells regulates CpG-mediated antibody production.

J Biol Chem 2008 Jan 9;283(2):802-8. Epub 2007 Oct 9.

Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angeles, California 90095, USA.

Hypomethylated CpG oligonucleotides (CpG) are not only potent adjuvants for enhancing adaptive immune responses but may also play a critical role in the development of autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). Here we provide evidence that, in addition to dendritic cells, murine B lymphocytes also exhibit a type I IFN response to CpG-B. Unlike dendritic cells, B cell-mediated type I IFN induction depended on the transcription factor IRF3, but similar to dendritic cells this pathway was independent of the IRF3 kinase TBK1. Utilizing type I IFN receptor-deficient mice, we were able to demonstrate that this IFN pathway enhanced Syndecan-1 expression and IgM production and was required for IgG2a production following CpG-B stimulation. Overall, our findings identify a unique IFN pathway in B cells that may play a central role in mediating B cell biology in response to CpG, potentially implicating this pathway in autoantibody production and the pathogenesis of certain autoimmune diseases.
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http://dx.doi.org/10.1074/jbc.M704755200DOI Listing
January 2008

TRAF3 and its biological function.

Adv Exp Med Biol 2007 ;597:48-59

Department of Microbiology, Immunology and Molecular Genetics, 8University of California, Los Angeles, Los Angeles, California 90095, USA.

Tumor necrosis factor receptor associated factor 3 (TRAF3) is one of the most enigmatic members in the TRAF family that consists of six members, TRAF1 to 6. Despite its similarities with other TRAFs in terms of structure and protein-protein association, overexpression of TRAF3 does not induce activation of the commonly known TRAF-inducible signaling pathways, namely NF-kappaB and JNK. This lack of a simple functional assay in combination with the mysterious early lethality of the TRAF3-deficient mice has made the study of the biological function of TRAF3 challenging for almost ten years. Excitingly, TRAF3 has been identified recently to perform two seemingly distinct roles. Namely, TRAF3 functions as a negative regulator of the NF-kappaB pathway and separately, as a positive regulator of type I IFN production, placing itself as a critical regulator of both innate and adaptive immune responses.
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http://dx.doi.org/10.1007/978-0-387-70630-6_4DOI Listing
August 2007

Specificity of TRAF3 in its negative regulation of the noncanonical NF-kappa B pathway.

J Biol Chem 2007 Feb 11;282(6):3688-94. Epub 2006 Dec 11.

Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California 90095, USA.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.
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http://dx.doi.org/10.1074/jbc.M610271200DOI Listing
February 2007

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency.

J Exp Med 2006 Oct 2;203(11):2413-8. Epub 2006 Oct 2.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.
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http://dx.doi.org/10.1084/jem.20061166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2118128PMC
October 2006

Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif.

EMBO J 2006 Jul 6;25(14):3257-63. Epub 2006 Jul 6.

Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA.

Upon recognition of viral infection, RIG-I and Helicard recruit a newly identified adapter termed Cardif, which induces type I interferon (IFN)-mediated antiviral responses through an unknown mechanism. Here, we demonstrate that TRAF3, like Cardif, is required for type I interferon production in response to intracellular double-stranded RNA. Cardif-mediated IFNalpha induction occurs through a direct interaction between the TRAF domain of TRAF3 and a TRAF-interaction motif (TIM) within Cardif. Interestingly, while the entire N-terminus of TRAF3 was functionally interchangeable with that of TRAF5, the TRAF domain of TRAF3 was not. Our data suggest that this distinction is due to an inability of the TRAF domain of TRAF5 to bind the TIM of Cardif. Finally, we show that preventing association of TRAF3 with this TIM by mutating two critical amino acids in the TRAF domain also abolishes TRAF3-dependent IFN production following viral infection. Thus, our findings suggest that the direct and specific interaction between the TRAF domain of TRAF3 and the TIM of Cardif is required for optimal Cardif-mediated antiviral responses.
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http://dx.doi.org/10.1038/sj.emboj.7601220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1523175PMC
July 2006

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response.

Nature 2006 Jan 23;439(7073):208-11. Epub 2005 Nov 23.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, 609 Charles E. Young Dr. East, Los Angeles, California 90095, USA.

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.
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http://dx.doi.org/10.1038/nature04374DOI Listing
January 2006

Type I interferon production enhances susceptibility to Listeria monocytogenes infection.

J Exp Med 2004 Aug 9;200(4):437-45. Epub 2004 Aug 9.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, 8-240 Factor Bldg., 10833 Le Conte Ave., 90095, USA.

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes-induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.
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http://dx.doi.org/10.1084/jem.20040712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211937PMC
August 2004

Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-kappaB activation pathways.

Proc Natl Acad Sci U S A 2004 May 17;101(21):8108-13. Epub 2004 May 17.

Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.

B lymphocytes can be activated by many different stimuli. However, the mechanisms responsible for the signaling and functional specificities of individual stimuli remain to be elucidated. Here, we have compared the contribution of the type 1 (p50-dependent) and type 2 (p52-dependent) NF-kappaB activation pathways to cell survival, proliferation, homotypic aggregation, and specific gene regulation of murine primary B lymphocytes. Whereas lipopolysaccharide (LPS) and B cell activation factor (BAFF) mainly activate the type 1 or type 2 pathways, respectively, CD40 ligand (CD40L) strongly activates both. Rescue of spontaneous apoptosis is diminished in p52(-/-) B cells after BAFF stimulation and in p50(-/-)c-Rel(-/-) B cells after LPS stimulation. Interestingly, significant CD40-induced B cell survival is still observed even in p50(-/-)c-Rel(-/-)p65(-/+) B cells, which is correlated with the ability of CD40L to up-regulate Bcl-x(L) expression in these cells. CD40L- and LPS-induced B cell proliferation, as well as up-regulation of proliferation-related genes, however, are greatly reduced in c-Rel(-/-) and p50(-/-)c-Rel(-/-) B cells but are normal in p52(-/-) B cells. We have further demonstrated that both c-Rel and p52 are required for CD40-mediated B cell homotypic aggregation, which explains well why neither LPS nor BAFF has this function. Overall, our studies suggest that both type 1 and type 2 NF-kappaB pathways contribute to the gene expression and biological program unique for CD40 in B cell activation.
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http://dx.doi.org/10.1073/pnas.0402629101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419565PMC
May 2004

Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes.

Proc Natl Acad Sci U S A 2002 Feb;99(3):1497-502

Molecular Biology Institute and Medical Scientist Training Program, School of Medicine, University of California, Los Angeles, CA 90095, USA.

CD40/CD40L interaction is essential for multiple biological events in T dependent humoral immune responses, including B cell survival and proliferation, germinal center and memory B cell formation, and antibody isotype switching and affinity maturation. By using high-density microarrays, we examined gene expression in primary mouse B lymphocytes after multiple time points of CD40L stimulation. In addition to genes involved in cell survival and growth, which are also induced by other mitogens such as lipopolysaccharide, CD40L specifically activated genes involved in germinal center formation and T cell costimulatory molecules that facilitate T dependent humoral immunity. Next, by examining the roles of individual CD40-activated signal transduction pathways, we dissected the overall CD40-mediated response into genes independently regulated by the individual pathways or collectively by all pathways. We also found that gene down-regulation is a significant part of the overall response and that the p38 pathway plays an important role in this process, whereas the NF-kappa B pathway is important for the up-regulation of primary response genes. Our finding of overlapping independent control of gene expression modules by different pathways suggests, in principle, that distinct biological behaviors that depend on distinct gene expression subsets can be manipulated by targeting specific signaling pathways.
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http://dx.doi.org/10.1073/pnas.032665099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122219PMC
February 2002