TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification.

Reciprocal gene activation and restriction during cell type differentiation from a common lineage is a hallmark of mammalian organogenesis. A key question, then, is whether a critical transcriptional activator of cell type-specific gene targets can also restrict expression of the same genes in other cell types. Here, we show that whereas the pituitary-specific POU domain factor Pit-1 activates growth hormone gene expression in one cell type, the somatotrope, it restricts its expression from a second cell type, the lactotrope. This distinction depends on a two-base pair spacing in accommodation of the bipartite POU domains on a conserved growth hormone promoter site. The allosteric effect on Pit-1, in combination with other DNA binding factors, results in the recruitment of a corepressor complex, including nuclear receptor corepressor N-CoR, which, unexpectedly, is required for active long-term repression of the growth hormone gene in lactotropes.

Structure of BamHI bound to nonspecific DNA: a model for DNA sliding.

The central problem faced by DNA binding proteins is how to select the correct DNA sequence from the sea of nonspecific sequences in a cell. The problem is particularly acute for bacterial restriction enzymes because cleavage at an incorrect DNA site could be lethal. To understand the basis of this selectivity, we report here the crystal structure of endonuclease BamHI bound to noncognate DNA. We show that, despite only a single base pair change in the recognition sequence, the enzyme adopts an open configuration that is on the pathway between free and specifically bound forms of the enzyme. Surprisingly, the DNA drops out of the binding cleft with a total loss of base-specific and backbone contacts. Taken together, the structure provides a remarkable snapshot of an enzyme poised for linear diffusion (rather than cleavage) along the DNA.

Crystallization of restriction endonuclease BamHI with nonspecific DNA.

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5'-ATGAATCCATA-3', yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA-protein complexes.

The role of metals in catalysis by the restriction endonuclease BamHI.

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI-DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.

Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase. Activity toward different substrates and effects of neighboring residues.

Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM beta-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme. The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type. Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK's for the wild type. This effect was predominantly due to changes in Km. In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km for several side chain-containing penicillin substrates increases when the pH is above 5.5. 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect. With benzylpenicillin, ampicillin, and carbenicillin, kcat for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp132; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells. The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp132 for the complex stability with side chain-containing substrates. The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme.

A new TEM beta-lactamase double mutant with broadened specificity reveals substrate-dependent functional interactions.

Using a random combinatorial mutagenesis of TEM beta-lactamase, directed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S. Additionally, by using cloning strategies and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E104K/G238S, which broaden the specificity of clinically isolated TEM beta-lactamase variants. The kinetic characterization of the purified double mutant E104M/G238S and its single counterparts E104M and G238S was carried out. The single mutant E104M exhibited increased kcat values against all substrates tested. Km values remained similar to the values shown by the wild-type enzyme. The mutation at E104M was responsible for the increased hydrolysis rate against cefuroxime shown by the double mutant E104/G238S. The effect of mutation G238S varied more pronouncedly, depending on the substrate. In general, a lower Km was observed, but also a decreased kcat. The double mutant E104M/G238S exhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations. We observed nearly a 1000-fold greater kcat/Km for the double mutant than for the wild type. This improvement in catalysis was the consequence of increased kcat and decreased Km values. Computed contact interactions from modeling substrate complexes show reliable results only for benzylpenicillin. The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants. Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the single and double mutants, showed that the quantitative effect of a second mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.