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PubMed1998

Mapping the sigma70 subunit contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease.

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several sigma subunits. To characterize the proximity between sigma70, the major sigma for transcription of the growth-related genes, and the core enzyme subunits (alpha2 beta beta'), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of sigma70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated sigma70 was bound to the core enzyme, which led to cleavage of nearby sites on the beta and beta' subunits (but not alpha). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71-75], the cut sites from different probe-modified sigma70 proteins are clustered in distinct regions of the subunits. On the beta subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the beta' subunit, the cleavage was identified within the sequence 228-461, including beta' conserved regions C and D (which comprise part of the catalytic center).

Bacterial Proteins↗
PubMed2003

Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures.

The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood. We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, respectively, were determined. 422aP2 gene expression was analyzed. Osteocalcin promoter activity was measured by cotransfecting the cells with the phOC-luc and pSV beta-Gal plasmids. Lovastatin enhanced osteoblast differentiation as assessed by a 1.8x increase in expression of Cbfa1/Runx2 and by a 5x increase in osteocalcin promoter activity. Expression of PPARgamma2 was decreased by 60%. By enhancing osteoblast gene expression and by inhibiting adipogenesis, lovastatin may shunt uncommitted osteoprogenitor cells in marrow from the adipocytic to the osteoblastic differentiation pathway. Future evaluation of lovastatin and other lipid-lowering drugs will help determine their potential as therapeutic agents for osteonecrosis and osteoporosis.

Adipocytes↗
PubMed2000

An initiator element mediates autologous downregulation of the human type A gamma -aminobutyric acid receptor beta 1 subunit gene.

The regulated expression of type A gamma-aminobutyric acid receptor (GABA(A)R) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABA(A)R subunit gene expression, including decreased beta1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABA(A)R number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABA(A)R beta1 subunit gene that is neuron specific and autologously down-regulated by GABA. beta1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABA(A)R activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3' flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of beta1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of beta1 subunit GABA(A)R gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.

Animals↗
PubMed1989

Refined structure of spinach glycolate oxidase at 2 A resolution.

The amino acid sequence of glycolate oxidase from spinach has been fitted to an electron density map of 2.0 A nominal resolution and the structure has been refined using the restrained parameter least-squares refinement of Hendrickson and Konnert. A final crystallographic R-factor of 18.9% was obtained for 32,888 independent reflections from 5.5 to 2 A resolution. The geometry of the model, consisting of 350 amino acid residues, the cofactor flavin mononucleotide and 298 solvent molecules, is close to ideal with root-mean-square deviations of 0.015 A in bond lengths and 2.6 degrees in bond angles. The expected trimodal distribution with preference for staggered conformation is obtained for the side-chain chi 1-angles. The core of the subunit is built up from the eight beta-strands in the beta/alpha-barrel. This core consists of two hydrophobic layers. One in the center is made up of residues pointing in from the beta-strands towards the barrel axis and the second, consisting of two segments of residues, pointing out from the beta-strands towards the eight alpha-helices of the barrel and pointing from the helices towards the strands. The hydrogen bond pattern for the beta-strands in the beta/alpha-barrel is described. There are a number of residues with 3(10)-helix conformation, in particular there is one left-handed helix. The ordered solvent molecules are organized mainly in clusters. The average isotropic temperature factor is quite high, 27.1 A2, perhaps a reflection of the high solvent content in the crystal. The octameric glycolate oxidase molecule, which has 422 symmetry, makes strong interactions around the 4-fold axis forming a tight tetramer, but only weak interactions between the two tetramers forming the octamer.

Alcohol Oxidoreductases↗
PubMed2004

Pathophysiology of vascular calcification in chronic kidney disease.

Patients with chronic kidney disease (CKD) on dialysis have 2- to 5-fold more coronary artery calcification than age-matched individuals with angiographically proven coronary artery disease. In addition to increased traditional risk factors, CKD patients also have a number of nontraditional cardiovascular risk factors that may play a prominent role in the pathogenesis of arterial calcification, including duration of dialysis and disorders of mineral metabolism. In histological specimens from the inferior epigastric artery of dialysis patients, we have found expression of the osteoblast differentiation factor core binding factor alpha-1 (Cbfa1) and several bone-associated proteins (osteopontin, bone sialoprotein, alkaline phosphatase, type I collagen) in both the intima and medial layers when calcification was present. In cultured vascular smooth muscle cells, the addition of pooled serum from dialysis patients (versus normal healthy controls) accelerated mineralization and increased expression of Cbfa1, osteopontin, and alkaline phosphatase to a similar magnitude as does beta-glycerophosphate alone. However, a lack of inhibitors of calcification may also be important. Dialysis patients with low levels of serum fetuin-A, a circulating inhibitor of mineralization, have increased coronary artery calcification and fetuin-A can inhibit mineralization of vascular smooth muscle cells in vitro. These data support that elevated levels of phosphorus and/or other potential uremic toxins may play an important role by transforming vascular smooth muscle cells into osteoblast-like cells, which can produce a matrix of bone collagen and noncollagenous proteins. This nidus can then mineralize if the balance of pro-mineralizing factors outweighs inhibitory factors.

Animals↗
PubMed1996

The solution structure of the bovine S100B protein dimer in the calcium-free state.

BACKGROUND: S100B (S100beta) is a member of the S100 family of small calcium-binding proteins: members of this family contain two helix-loop-helix calcium-binding motifs and interact with a wide range of proteins involved mainly in the cytoskeleton and cell proliferation. S100B is a neurite-extension factor and levels of S100B are elevated in the brains of patients with Alzheimer's disease or Down's syndrome: the pattern of S100B overexpression in Alzheimer's disease correlates with the pattern of neuritic-plaque formation. Identification of a growing class of S100 proteins and the likely neurochemical importance of S100B make the determination of the structure of S100B of interest. RESULTS: We have used NMR to determine the structure of the reduced S100B homodimer in the absence of calcium. Each monomer consists of a four-helix bundle, arranged in the dimer in an antiparallel fashion. The fourth helix of each monomer runs close to the equivalent helix of the other monomer for almost its full length, extending the hydrophobic core through the interface. The N-terminal, but not the C-terminal, calcium-binding loop is similar to the equivalent loop in the monomeric S100 protein calbindin and is in a conformation ready to bind calcium. CONCLUSIONS: The novel dimer structure reported previously for calcyclin (S100A6) is the common fold for the dimeric S100B proteins. Calcium binding to the C-terminal calcium-binding loop would be expected to require a conformational change, which might provide a signal for activation. The structure suggests regions of the molecule likely to be involved in interactions with effector molecules.

Amino Acid Sequence↗
PubMed2002

Astrocytic activation and delayed infarct expansion after permanent focal ischemia in rats. Part I: enhanced astrocytic synthesis of s-100beta in the periinfarct area precedes delayed infarct expansion.

An astrocytic protein S-100beta enhances the expression of inducible nitric oxide synthase in cultured astrocytes at micromolar concentrations, leading to nitric oxide-mediated death of cocultured neurons. The present study examined whether S-100beta production by reactive astrocytes accumulating within the periinfarct area was related to delayed expansion of infarct volume after permanent middle cerebral artery occlusion in the rat. After rapid increases during the initial 24 hours, the increase of infarct volume then decelerated while maintaining the increasing tendency until 168 hours in this model, attaining a significant difference compared with that at 24 hours. In the periinfarct area, the number of reactive astrocytes expressing both S-100 and glial fibrillary acidic protein, the tissue level of S-100beta as measured by the sandwich enzyme-linked immunosolvent assay method using anti-S-100beta monoclonal antibody, and the number of terminal deoxynucleotidyl transferase-mediated 2;-deoxyuridine 5;-triphosphate-biotin nick end labeling-positive cells were significantly increased preceding the delayed expansion of infarct volume. The CSF concentration of S-100beta showed a biphasic increase, presumably reflecting the immediate release from astrocytes within the ischemic core and the subsequent production in reactive astrocytes within the periinfarct area. These results show for the first time that the enhanced synthesis of S-100beta by reactive astrocytes participates in the inflammatory responses within the periinfarct area, which may be related to the occurrence of delayed infarct expansion as a major component of the cytokine network.

Animals↗
PubMed1996

Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. An alpha/beta hydrolase structure that is different from the alpha/beta hydrolase fold.

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

Amino Acid Sequence↗
PubMed2005

Interplay between interferon and other cytokine systems in bone metabolism.

Interferons (IFNs) play crucial roles in the regulation of a wide variety of innate and adaptive immune responses. Type I interferons (IFN-alpha/beta) are central to the host defense against pathogens such as viruses, whereas type II interferon (IFN-gamma) mainly contributes to the T-cell-mediated regulation of the immune responses. Studies of bone destruction associated with rheumatoid arthritis have highlighted the importance of the interaction between the immune and skeletal systems. Recently, a new research area, termed osteoimmunology, has been spawned by a series of studies focusing on the signaling networks between IFN and other cytokines in bone metabolisms. It has been revealed that IFN-gamma interferes with the osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand (RANKL), and this mechanism is critical for the suppression of pathological bone resorption associated with inflammation. In addition, RANKL induces the IFN-beta gene in osteoclast precursor cells, and this induction constitutes a critical aspect of the negative feedback regulation mechanisms of RANKL signaling to suppress excessive osteoclastogenesis. Furthermore, a novel function of signal transducer and activator of transcription 1 (Stat1), the essential transcription factor for both type I and type II IFN responses, was revealed in the regulation of osteoblast differentiation. Collectively, these studies unveil novel aspects of the IFN system and indicate the operation of the intricate signaling network among IFN and other cytokine systems in bone remodeling, which might offer a molecular basis for the treatment of bone diseases.

Animals↗
PubMed2003

IL-4 suppresses the expression and the replication of hepatitis B virus in the hepatocellular carcinoma cell line Hep3B.

IL-4 has been known as a Th2 cytokine and can act on B cells, T cells, and monocytes. In this study we demonstrate that IL-4Rs are expressed on human hepatocellular carcinoma (HCC) cells. We found that IL-4 suppresses hepatitis B surface Ag (HBsAg) mRNA and HBsAg production in the Hep3B cell line, which contains an integrated hepatitis B virus (HBV) genome and constitutively secretes HBsAg. When Hep3B cells are further transfected with the plasmid pHBV3.6 that contains >1 U of HBV genome, IL-4 could suppress the production of all HBV RNA and secreted HBsAg and hepatitis B virus e Ag. Furthermore, an endogenous DNA polymerase activity assay shows a decrease in HBV DNA after IL-4 treatment. Using luciferase reporter assays we have demonstrated that IL-4 could suppress the activity of the surface promoter II and the core promotor (CP). To delineate how IL-4 suppressed the transcription of HBV genes, we have examined the effect of IL-4 on the expression of transcription factors that are known to bind to the core upstream regulatory sequence, which colocalizes with enhancer II of the HBV genome. Our results demonstrate that IL-4 suppresses the expression of C/EBPalpha. Furthermore, overexpression of C/EBPalpha blocked 43 and 30% of the IL-4-mediated suppression of CP activity and IL-4-induced suppression of pregenomic RNA, respectively. Finally, we have demonstrated that mutations affecting the C/EBPalpha-binding sites on core upstream regulatory sequence/enhancer II completely abolish the IL-4-mediated suppression of CP activity. Thus, down-regulation of C/EBPalpha may be involved in the anti-HBV effect of IL-4 in Hep3B cells.

Antiviral Agents↗
PubMed1991

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

Amino Acid Sequence↗
PubMed2004

Structural and functional characterization on the interaction of yeast TFIID subunit TAF1 with TATA-binding protein.

General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively). Our NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region.

Amino Acid Sequence↗
PubMedDate not supplied

The cloned promoter of the human DNA beta-polymerase gene contains a cAMP response element functional in HeLa cells.

The mammalian DNA beta-polymerase (beta-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but is expressed in rodents in a tissue-specific fashion. As revealed by transient expression experiments with wild-type and mutated beta-pol promoter fusion genes, the cloned human beta-pol promoter is transcriptionally regulated by signals acting through the single palindromic sequence (GT-GACGTCAC) known as an ATF/CRE-binding site centered at position -45 in the core promoter. Although the mere presence of the ATF/CRE palindromic sequence in a promoter does not always confer cAMP responsiveness or protein binding over and around the ATF/CRE sequence, we find that agents that increase cAMP levels (forskolin and IBMX) in HeLa cells activate the beta-pol promoter; activation also can be observed by coexpression of the protein kinase A catalytic subunit. Experiments with mutagenized beta-pol promoters indicate that the ATF/CRE-binding site mediates these effects. Thus, the ATF/CRE-binding site in the context of this TATA-less constitutive promoter is able to respond to the kinase A signal transduction pathway.

Base Sequence↗
PubMed2001

TATA binding protein-associated CK2 transduces DNA damage signals to the RNA polymerase III transcriptional machinery.

Here we report that RNA polymerase (pol) III transcription is repressed in response to DNA damage by downregulation of TFIIIB, the core component of the pol III transcriptional machinery. Protein kinase CK2 transduces this stress signal to TFIIIB. CK2 associates with and normally activates the TATA binding protein (TBP) subunit of TFIIIB. The beta regulatory subunit of CK2 binds to TBP and is required for high TBP-associated CK2 activity and pol III transcription in unstressed cells. Transcriptional repression induced by DNA damage requires CK2 and coincides with downregulation of TBP-associated CK2 and dissociation of catalytic subunits from TBP-CK2 complexes. Therefore, CK2 is the terminal effector in a signaling pathway that represses pol III transcription when genome integrity is compromised.

Casein Kinase II↗
PubMed1999

Crystal structure of a transcriptionally active Smad4 fragment.

BACKGROUND: Smad4 functions as a common mediator of transforming growth factor beta (TGF-beta) signaling by forming complexes with the phosphorylated state of pathway-restricted SMAD proteins that act in specific signaling pathways to activate transcription. SMAD proteins comprise two domains, the MH1 and MH2 domain, separated by a linker region. The transcriptional activity and synergistic effect of Smad4 require a stretch of proline-rich sequence, the SMAD-activation domain (SAD), located N-terminal of the MH2 domain. To understand how the SAD contributes to Smad4 function, the crystal structure of a fragment including the SAD and MH2 domain (S4AF) was determined. RESULTS: The structure of the S4AF trimer reveals novel features important for Smad4 function. A Smad4-specific sequence insertion within the MH2 domain interacts with the C-terminal tail to form a structural extension from the core. This extension (the TOWER) contains a solvent-accessible glutamine-rich helix. The SAD reinforces the TOWER and the structural core through interactions; two residues involved in these interactions are targets of tumorigenic mutation. The solvent-accessible proline residues of the SAD are located on the same face as the glutamine-rich helix of the TOWER, forming a potential transcription activation surface. A tandem sulfate-ion-binding site was identified within the subunit interface, which may interact with the phosphorylated C-terminal sequence of pathway-restricted SMAD proteins. CONCLUSIONS: The structure suggests that the SAD provides transcriptional capability by reinforcing the structural core and coordinating with the TOWER to present the proline-rich and glutamine-rich surfaces for interaction with transcription partners. The sulfate-ion-binding sites are potential 'receptors' for the phosphorylated sequence of pathway-restricted SMAD proteins in forming a heteromeric complex. The structure thus provides a new model that can be tested using biochemical and cellular approaches.

Amino Acid Sequence↗
PubMed2006

Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion.

Type IV pili are long, flexible filaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin subunits. This review focuses on the structural information available for each pilin subclass, type IVa and type IVb, highlighting the contributions crystal and nuclear magnetic resonance structures have made in understanding pilus function and assembly. In addition, the type II secretion pseudopilus subunit structure and helical assembly is compared to that of the type IV pilus. The pilin subunits adopt an alphabeta-roll fold formed by the hydrophobic packing of the C-terminal half of a long alpha-helix against an antiparallel beta-sheet. The conserved N-terminal half of the same alpha-helix, as well as two sequence- and structurally-variable regions, protrude from this globular head domain. Filament models have a hydrophobic core formed by the signature long alpha-helices, with variable regions at the filament surface.

Amino Acid Sequence↗
PubMed1999

Solution properties of the free and DNA-bound Runt domain of AML1.

The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.

Base Sequence↗
PubMed2002

Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation.

It is well established that p300 plays an important role in mediating gene expressions. However, it is less clear how its binding is influenced by physiological stimuli and how its altered binding affects transactivator acetylation and binding. In this study, we determined p300 binding to a core cyclooxygenase-2 (COX-2) promoter region by chromatin immunoprecipitation and streptavidin-agarose pull-down assays in basal and tumor necrosis factor-alpha (TNFalpha)-treated human foreskin fibroblasts. We found basal binding of p300, p50/p65 NF-kappaB, cyclic AMP regulatory element-binding protein-2, CCAAT/enhancer-binding protein beta, and c-Jun. p50/p65 and p300 binding was selectively increased by TNFalpha. Immunoprecipitation confirmed direct interaction of p300 with NF-kappaB and the other involved transactivators. p50 acetylation was detected in resting cells and was increased by TNFalpha or lipopolysaccharide. Overexpression of p300 augmented p50 acetylation, which was attenuated by deletion of its histone acetyltransferase domain. Enhanced p50 acetylation correlated with increased p50 binding to COX-2 promoter and transcriptional activation. Co-transfection of E1A with p300 abrogated p50 acetylation and p50 binding. These findings suggest that up-regulation of p300 binding and its acetylation of NF-kappaB occupies a central position in COX-2 promoter activation.

Acetylation↗