NF-kappaB signaling pathways in mammalian and insect innate immunity.
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Animals↗
Biomedical subjects
T Maniatis
Publications and source records attributed to T Maniatis.
A new family of IKK-related kinases may function as I kappa B kinase kinases.
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Adaptor Proteins, Signal Transducing↗
A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
The transcriptional coactivators CBP and p300 are critical regulators of metazoan gene expression. They associate with many different DNA-bound transcription factors through small, conserved domains. We have identified a compactly folded 46 residue domain in CBP and p300, the IRF-3 binding domain (IBiD), and we have determined its structure by NMR. It has a helical framework containing an apparently flexible polyglutamine loop that participates in ligand binding. Spectroscopic data indicate that induced folding accompanies association of IBiD with its partners, which exhibit no evident sequence similarities. We demonstrate the significance both in vitro and in vivo of interactions between IBiD and a number of diverse partners. Thus, IBiD is an important contributor to signal integration by CBP and p300.
Amino Acid Sequence↗
The role of U2AF35 and U2AF65 in enhancer-dependent splicing.
Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.
Base Sequence↗
Comparative DNA sequence analysis of mouse and human protocadherin gene clusters.
The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.
Animals↗
Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter.
Here, we show that the IFN-beta enhanceosome activates transcription by directing the ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. The enhanceosome is assembled in the nucleosome-free enhancer region of the IFN-beta gene, leading to the modification and remodeling of a strategically positioned nucleosome that masks the TATA box and the start site of transcription. Initially, the GCN5 complex is recruited, which acetylates the nucleosome, and this is followed by recruitment of the CBP-PolII holoenzyme complex. Nucleosome acetylation in turn facilitates SWI/SNF recruitment by CBP, resulting in chromatin remodeling. This program of recruitment culminates in the binding of TFIID to the promoter and the activation of transcription.
Acetylation↗
A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity.
Here we report the identification of a Drosophila IkappaB kinase complex containing DmIKKbeta and DmIKKgamma, homologs of the human IKKbeta and IKKgamma proteins. We show that this complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, we find that the activated DmIKK complex, as well as recombinant DmIKKbeta, can phosphorylate Relish in vitro. Thus, we propose that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway.
Animals↗
Large exons encoding multiple ectodomains are a characteristic feature of protocadherin genes.
Recent studies revealed a striking difference in the genomic organization of classic cadherin genes and one family of "nonclassic cadherin" genes designated protocadherins. Specifically, the DNA sequences encoding the ectodomain repeats of classic cadherins are interrupted by multiple introns. By contrast, all of the encoded ectodomains of each member of the protocadherin gene clusters are present in one large exon. To determine whether large ectodomain exons are a general feature of protocadherin genes we have investigated the genomic organization of several additional human protocadherin genes by using DNA sequence information in GenBank. These genes include protocadherin 12 (Pcdh12), an ortholog of the mouse vascular endothelial cadherin-2 gene; hFmi1 and hFmi2, homologs of the Drosophila planar cell polarity gene, flamingo; hFat2, a homolog of the Drosophila tumor suppressor gene fat; and the Drosophila DN-cadherin and DE-cadherin genes. Each of these genes was found to be a member of the protocadherin subfamily, based on amino acid sequence comparisons of their ectodomains. Remarkably, all of these protocadherin genes share a common feature: most of the genomic DNA sequences encoding their ectodomains are not interrupted by an intron. We conclude that the presence of unusually large exons is a characteristic feature of protocadherin genes.
Amino Acid Sequence↗
Role of adenine functional groups in the recognition of the 3'-splice-site AG during the second step of pre-mRNA splicing.
The AG dinucleotide at the 3' splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG --> GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG --> CG) or uracil (AG --> UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG --> HG). When adenine was replaced by a purine base (AG --> PG) or by 7-deazaadenine (AG --> c(7)AG), little effect on the second step was observed, suggesting that the 6-NH(2) and N(7) groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG --> 2-APG) had no effect on the second step. Taken together, our results suggest that the N(1) group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.
Adenine↗
IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex.
Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.
Amino Acid Sequence↗
Assembly of a functional beta interferon enhanceosome is dependent on ATF-2-c-jun heterodimer orientation.
Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2-c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-beta) gene. Here we report that within the IFN-beta enhanceosome the ATF-2-c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2-c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2-c-jun binding site is required for virus induction of the IFN-beta gene and for IRF-3-dependent activation of a composite ATF-2- c-jun-IRF site in the IFN-beta promoter. We also show that in vitro the DNA-bound ATF-2-c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-beta enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-beta reporter constructs reveal that recruitment of IRF-3 to the IFN-beta promoter upon virus infection is dependent on the orientation of the ATF-2-c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-beta enhanceosome.
Activating Transcription Factor 2↗
Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter.
The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.
DNA-Binding Proteins↗
A striking organization of a large family of human neural cadherin-like cell adhesion genes.
We have identified 52 novel human cadherin-like genes organized into three closely linked clusters. Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.
Amino Acid Sequence↗
Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing.
Two distinct functions have been proposed for the serine-arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein-protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.
Exons↗
A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways.
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Animals↗
SR proteins are 'locators' of the RNA splicing machinery.
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Alternative Splicing↗
PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins.
The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of beta-interferon (IFN-beta) and c-myc gene expression. Here, we show that PRDI-BF1 represses transcription of the IFN-beta promoter and of an artificial promoter through an active repression mechanism. We also identified a minimal repression domain in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein. Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors. In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain. We also find that the amino-terminal glutamine-rich domains of hGrg and TLE1 are sufficient to mediate dimerization of the two Groucho family proteins. Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-beta promoter activity after virus induction. We conclude that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and suggest that such corepressor complexes are required for the postinduction repression of the IFN-beta promoter.
Amino Acid Sequence↗
Virus infection leads to localized hyperacetylation of histones H3 and H4 at the IFN-beta promoter.
Transcriptional activation of the human interferon-beta (IFN-beta) gene by virus infection requires the assembly of a higher order nucleoprotein complex, the enhanceosome, which consists of the transcriptional activators NF-kappa B (p50/p65), ATF-2/c-jun, IRF-3 and IRF-7, architectural protein HMGI(Y), and the coactivators p300 and CBP. In this report, we show that virus infection of cells results in a dramatic hyperacetylation of histones H3 and H4 that is localized to the IFN-beta promoter. Furthermore, expressing a truncated version of IRF-3, which lacks a p300/CBP interaction domain, suppresses both histone hyperacetylation and activation of the IFN-beta gene. Thus, coactivator-mediated localized hyperacetylation of histones may play a crucial role in inducible gene expression.
Acetylation↗