DNA Consensus Site Research Articles

Exposure of Microglia to Interleukin-4 Represses NF-κB-Dependent Transcription of Toll-Like Receptor-Induced Cytokines.

Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas.

Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation.

Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin‐regulating antisense RNA (micF). In bacteria, σ 70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ 70 form a holoenzyme that recognizes ‐35 and ‐10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP‐σ 70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator‐specific features in activator binding and uncovers distinct roles of the two C‐terminal domains of RNAP α subunit.

Sensitive surface plasmon resonance detection of methyltransferase activity and screening of its inhibitors amplified by p53 protein bound to methylation-specific ds-DNA consensus sites

Abnormal DNA methylation is closely related to cancer initiation and progression, and strategies to assay methyltransferase activity and screen its inhibitors are essential for cancer diagnosis and therapy. In this work, surface plasmon resonance (SPR)-based assay for real-time and sensitive monitoring of DNA methyltransferase activity and screening of its inhibitors was conducted via methylation of double-stranded (ds)-DNA consensus sites and the follow-up p53 protein recognition. The consensus ds-DNA possesses a specific sequence of 5′-CCGG-3′ in which the second C base can be methylated by M.SssI methyltransferase (M.SssI MTase) and the methylation process impedes the recognition and cleavage of the ds-DNA by HpaII endonuclease, thus, the attachment of p53 protein leads to remarkable SPR signals. In contrast, inhibition of M.SssI MTase activity by a potent inhibitor leaves the consensus ds-DNA unmethylated, and the cleavage of the ds-DNA by HpaII prevents p53 protein from adsorbing onto the chip surface, leading to tiny SPR signals. The binding affinity (KD) between p53 protein and the methylated consensus ds-DNA was deduced to be 3.04 nM, evidencing the strong binding capability. Two nucleoside inhibitors of 5-Azacytidine (5-Aza) and 5-aza-2′-deoxycytidine (5-Aza-dC), and a non-nucleoside inhibitor of procaine were examined, and their half-maximal inhibiting concentration (IC50) values were highly comparable with those by other methods. The sensing protocol has been successfully utilized for the assay of M.SssI MTase activity in normal and cancer cell lysates. The proof-of-concept experiments demonstrate that SPR serves as a viable means for sensitive detection of methyltransferase activity and screening of its inhibitors using p53 protein bound to methylation-specific ds-DNA consensus sites.

Structure of the Forkhead Domain of FOXA2 Bound to a Complete DNA Consensus Site.

FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). The FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and fewer minor groove contacts in the FOXA2 structure than in the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.

Abstract 2207: Elucidating mechanisms employed by NFIL3 to impact cancer cell survival

Abstract NFIL3 was recently identified as a novel modulator of FOXO1 output on the PI3K pathway. NFIL3 bound to DNA to physically block FOXO1 recruitment to genes that induce apoptosis. The diminishment in NFIL3 expression via RNA interference led to FOXO recruitment to cell death genes, an increase in Histone H3 acetylation on these genes, and increased expression of these genes. However, a number of observations suggest that FOXO1 is not the main target of NFIL3 in these cells. First, the NFIL3 and FOXO1 DNA consensus sites have almost no similarity. Second, the loss of FOXO1 or all three FOXO factors failed to suppress the cell death phenotype upon the diminishment of NFIL3 in MEFs. The goal of this project is to identify the factor(s) that NFIL3 directly blocks from DNA recruitment to promote cellular survival. In previous work, the NFIL3 and FOXO1 sites were mapped as being adjacent on the DNA. The mapped NFIL3 site was required for NFIL3 to associate with the DNA in biochemical experiments and for NFIL3 to regulate the promoter sequence in reporter assays. The mapped NFIL3 site is a perfect match for the bZIP transcription factor CCAAT/enhancer binding protein beta (CEBPB) consensus sequence. NFIL3 has been shown to block the ability of CEBPB to activate transcription by competing for access to the same DNA consensus site in numerous contexts including neuronal regeneration, PKA pathway transcriptional programs and the regulation of hepatitis C viral genes. Aside from DBP in circadian rhythm, CEBPB is the most commonly antagonized factor by NFIL3 in the literature. We are investigating if NFIL3 hinders FOXO1 recruitment to cell death genes by directly antagonizing CEBPB recruitment to these genes using cell biological and bioinformatics approaches. We have found that CEBPB is required for efficient induction of a subset of NFIL3 repressed genes. Citation Format: Megan Keniry, Omar Caballero, Itzel Flores, Andrea Salinas. Elucidating mechanisms employed by NFIL3 to impact cancer cell survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2207. doi:10.1158/1538-7445.AM2015-2207

The effect of resveratrol and its methylthio-derivatives on EGFR and Stat3 activation in human HaCaT and A431 cells

Epidermal growth factor receptor (EGFR) interacting with Stat3 is considered to be an attractive therapeutic target. In the current study, we investigated the effect of resveratrol and its two 4′-methylthio-trans-stilbene derivatives (3-M-4′-MTS; S2) (3,5-DM-4″-MTS; S5) on EGFR and Stat3 activation in human immortalized HaCaT keratinocytes and epidermoid carcinoma A431 cells. In the HaCaT cells both derivatives, similarly as resveratrol, decreased the total level of the EGFR receptor. In the A431 cells, resveratrol in the higher dose significantly (p < 0.05) reduced Y1173 and Y1068 EGFR residue phosphorylation, while S2 affected only the phosphorylation of the Y1068 residue. In this cell line, resveratrol in both tested doses and the S2 derivative in the lower concentration significantly diminished Stat3 binding capacity to the DNA consensus site. The effect of the tested compounds on Stat3 activation in HaCaT cells was only slightly affected. These results indicate that methylthiostilbenes are not more potent modulators of the EGFR/Stat3 complex than resveratrol and that introducing an additional methoxy group makes them less effective.

Minimal NF-κB activity in neurons

Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, electrophoretic mobility shift assay (EMSA) of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-κB-regulated gene expression, and enzyme-linked immunosorbent assay (ELISA) of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-κB activity was nearly absent in neurons. Induced activity was minimal—many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-beta (IL-β). Neuronal NF-κB was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-κB activity.

Physical Interaction of the Proto-Oncogene EVI1 with Hypermethylated in Cancer 1 (HIC1) Deregulates EVI1 Mediated Apoptosis in Cancer Cells

Abstract 2449The ecotropic viral integration site I (EVII) gene has attracted considerable attention in recent years because of its reported involvement in chronic myeloid leukemia, high risk myelodysplastic syndrome and a very high percentage of acute myeloid leukemias. The most recent World Health Organization classification has designated AML with inv (3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. Studies also indicate that EVI1 may be overexpressed in a subset of human colon cancers, and that EVI1 might affect disease progression and/or sensitivity to chemotherapy. The presence of zinc finger domains that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, few target genes of EVI1 have been functionally identified. In the present study, we demonstrate by co-immunoprecipitation assay that the proximal zinc finger domain of EVI1 binds to HIC1, which abolishes the ability of EVI1 to induce expression of Bcl-xL. HIC1, is an epigenetically regulated transcriptional repressor that functionally cooperates with p53 to suppress age dependent development of cancer in mice. Inactivation of HIC1 results in up regulation of SIRT1, which deacetylate and inactivates p53; allowing cells to bypass apoptosis and DNA damage survival., Our study also demonstrates via indirect immuno fluorescence analysis that EVI1 and HIC1 proteins are colocalized in speckles in the nucleus. We recently reported that EVI1 not only binds the Bcl-xL promoter but also up regulates its transcriptional activity and protein expression. We now demonstrate by EMSA and luciferase assays that physical interaction between EVI1 and HIC1 releases the binding of EVI1 from the Bcl-xL promoter, and hence promotes apoptosis. Thus by directly binding to EVI1, HIC1 displaces EVI1 from its DNA consensus site leading to down regulation of Bcl-xL. Also we observed that posttranslational modification of EVI1, mainly acetylation plays a critical role in this EVI1-HIC1 protein-protein interaction. Further elucidation of the counteracting effects of HIC1 and EVI1 on Bcl-xL expression may provide new insights in EVI1 mediated disease progression. Disclosures:No relevant conflicts of interest to declare.

Elk-1 a Transcription Factor with Multiple Facets in the Brain

The ternary complex factor (TCF) Elk-1 is a transcription factor that regulates immediate early gene (IEG) expression via the serum response element (SRE) DNA consensus site. Elk-1 is associated with a dimer of serum response factor (SRF) at the SRE site, and its phosphorylation occurs at specific residues in response to mitogen-activated protein kinases (MAPKs), including c-Jun-N terminal kinase (JNK), p38/MAPK, and extracellular-signal regulated kinase (ERK). This phosphorylation event is critical for triggering SRE-dependent transcription. Although MAPKs are fundamental actors for the instatement and maintenance of memory, and much investigation of their downstream signaling partners have been conducted, no data yet clearly implicate Elk-1 in these processes. This is partly due to the complexity of Elk-1 sub-cellular localization, and hence functions, within neurons. Elk-1 is present in its resting state in the cytoplasm, where it colocalizes with mitochondrial proteins or microtubules. In this particular sub-cellular compartment, overexpression of Elk-1 is toxic for neuronal cells. When phosphorylated by the MAPK/ERK, Elk-1 translocates to the nucleus where it is implicated in regulating chromatin remodeling, SRE-dependent transcription, and neuronal differentiation. Another post-translational modification is the conjugation to SUMO (Small Ubiquitin-like MOdifier), which relocalizes Elk-1 in the cytoplasm. Thus, Elk-1 plays a dual role in neuronal functions: pro-apoptotic within the cytoplasm, and pro-differentiation within the nucleus. To address the role of Elk-1 in the brain, one must be aware of its multiple facets, and design molecular tools that will shut down Elk-1 expression, trafficking, or activation, in specific neuronal compartments. We summarize in this review the known molecular functions of Elk-1, its regulation in neuronal cells, and present evidence of its possible implication in model systems of synaptic plasticity, learning, but also in neurodegenerative diseases.

Foxa1 Functions as a Pioneer Transcription Factor at Transposable Elements to Activate Afp during Differentiation of Embryonic Stem Cells

Epigenetic control of genes that are silent in embryonic stem cells, but destined for expression during differentiation, includes distinctive hallmarks, such as simultaneous activating/repressing (bivalent) modifications of chromatin and DNA hypomethylation at enhancers of gene expression. Although alpha-fetoprotein (Afp) falls into this class of genes, as it is silent in pluripotent stem cells and activated during differentiation of endoderm, we find that Afp chromatin lacks bivalent histone modifications. However, critical regulatory sites for Afp activation, overlapping Foxa1/p53/Smad-binding elements, are located within a 300-bp region lacking DNA methylation, due to transposed elements underrepresented in CpG sequences: a short interspersed transposable element and a medium reiterated sequence 1 element. Forkhead family member Foxa1 is activated by retinoic acid treatment of embryonic stem cells, binds its DNA consensus site within the short interspersed transposable/medium reiterated sequence 1 elements, and displaces linker histone H1 from silent Afp chromatin. Small interfering RNA depletion of Foxa1 showed that Foxa1 is essential in providing chromatin access to transforming growth factor beta-activated Smad2 and Smad4 and their subsequent DNA binding. Together these transcription factors establish highly acetylated chromatin and promote expression of Afp. Foxa1 acts as a pioneer transcription factor in de novo activation of Afp, by exploiting a lack of methylation at juxtaposed transposed elements, to bind and poise chromatin for intersection with transforming growth factor beta signaling during differentiation of embryonic stem cells.

ZAC1 Is Up-regulated by Hypertonicity and Decreases Sorbitol Dehydrogenase Expression, Allowing Accumulation of Sorbitol in Kidney Cells

Affymetrix GeneChip technology was employed to detect differentially expressed genes in inner medullary collecting duct (IMCD3) cells grown under isotonic and hypertonic conditions. A marked up-regulation was found for the zinc-finger protein ZAC1 under hypertonic stress (219-fold, p < 0.001). Changes in expression for ZAC1 were verified by quantitative PCR for message and Western blotting for protein. In mouse and human kidney tissues, ZAC1 expression was substantial in the papilla and was absent in the cortex. Furthermore, ZAC1 expression significantly increased in the papilla of mice following 36 h of fluid restriction and decreased in polyuric mice consuming sucrose in water. Because ZAC1 has been described to be a potential negative regulator of sorbitol dehydrogenase (SDH) in hippocampal cells, we examined whether this relationship also occurs in kidney cells under hypertonic stress. We found that stable IMCD3 clones silenced for ZAC1 to varying levels demonstrated an inverse effect on SDH expression. ZAC1 binds to a consensus repression site within the promoter of SDH, pointing to a mechanism whereby ZAC1 acts by repressing SDH transcriptional activity during hypertonic conditions. Taken together, these data strongly suggest that ZAC1 is up-regulated under hypertonic stress and negatively regulates expression of SDH, allowing for accumulation of sorbitol as a compatible organic osmolyte.

Pathogenic mechanisms of tooth agenesis linked to paired domain mutations in human PAX9

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.

Regulation of the Transcription Factor YY1 in Mitosis

YY1 is a multifunctional protein involved in the regulation of basic cellular processes at the DNA, RNA, and protein levels. The link between YY1 and cell cycle regulation has long been proposed. YY1 has been shown to interact with, and regulate, Rb, p53, c‐Myc, and many other cell cycle regulatory proteins. In addition, YY1 plays a role in the up‐regulation of many genes needed at the G2/M transition of the cell cycle. On the other hand, YY1 up‐regulates genes at the exit from mitosis, at the M/G1 transition. The subcellular localization of YY1 has been previously investigated at the G1/S transition of the cell cycle. In this study, the regulation and localization of YY1 during mitosis have been examined. Using confocal microscopy imaging of HeLa cells, YY1 was found to be primarily nuclear at the G2/M transition, but rapidly distributes to the cytoplasm, away from the condensed chromosomes, during prophase. YY1 remains excluded from DNA throughout pro‐metaphase, metaphase, and anaphase. At telophase, YY1 was rapidly recruited back to the decondensing chromosomes. The YY1 protein levels do not change significantly during the cell cycle; however, the YY1 protein in HeLa whole cell extracts was unable to bind its DNA consensus site in an in vitro electrophoretic mobility shift assay. The possibility of posttranslational modification of YY1 during mitosis is the focus of this study. Funded by FSU College of Medicine.

Blimp-1Δexon7: A naturally occurring Blimp-1 deletion mutant with auto-regulatory potential

Blimp-1 is a master regulator of terminal B cell differentiation and plays a pivotal role in various developmental processes. In addition to full length Blimp-1, a Blimp-1 mRNA lacking exon 7 (Blimp-1Δ7) has been described to occur in murine B cells. The activity and function of the mutant mRNA-encoded protein (Blimp-1Δ7), lacking three crucial zinc fingers necessary for DNA interaction, is completely unknown. Since isoforms of other prdm family proteins affect each other's functions, we wondered whether Blimp-1Δ7 still plays a role in B cells, independent of direct DNA binding. In this study, we found that Blimp-1Δ7 is preferentially expressed in naïve CD19 + B cells. A fraction of Blimp-1Δ7 migrates to the nucleus, colocalizes with HDAC2 and is found at sites of repressed chromatin, although it does not bind to the Blimp-1 DNA consensus site. Unexpectedly, Blimp-1 and Blimp-1Δ7 homodimerize as well as heterodimerize with each other. Ectopic expression of Blimp-1Δ7 in WEHI 231 cells, a Blimp-1-negative murine lymphoma line, leads to cessation of proliferation and enhancement of apoptosis. Importantly, LPS-induced differentiation is suppressed in the presence of Blimp-1Δ7. This is in agreement with our finding that Blimp-1Δ7 interferes with endogenous Blimp-1 expression. Thus, our data suggest an auto-regulatory mechanism of Blimp-1 activation.

Mechanism of DNA-binding loss upon single-point mutation in p53

Over 50% of all human cancers involve p53 mutations,which occur mostly in the sequence-specific DNA-binding central domain (p53c), yielding little/non-detectable af?nity to the DNA consensus site. Despite our current understanding of protein-DNA recognition,the mechanism(s) underlying the loss in protein-DNA binding afnity/ specificity upon single-point mutation are not well understood. Our goal is to identify the common factors governing the DNA-binding loss of p53c upon substitution of Arg 273 to His or Cys,which are abundant in human tumours. By computing the free energies of wild-type and mutant p53c binding to DNA and decomposing them into contributions from individual residues, the DNA-binding loss upon charge/noncharge -conserving mutation of Arg 273 was attributed not only to the loss of DNA phosphate contacts, but also to longer-range structural changes caused by the loss of the Asp 281 salt-bridge. The results herein and in previous works suggest that Asp 281 plays a critical role in the sequence-specific DNA-binding function of p53c by (i)orienting Arg 273 and Arg 280 in an optimal position to interact with the phosphate and base groups of the consensus DNA, respectively, and (ii) helping to maintain the proper DNA-binding protein conformation.

Structural Basis for p53 Binding-induced DNA Bending

Specific p53 binding-induced DNA bending has important biological implications such as transcription activation. However, the detailed structures of the bent DNA and the p53-DNA complex are still unavailable, hampering our understanding of the mechanism for p53-induced DNA bending and its consequent biological significance. To gain insight into the p53 binding-induced DNA bending, we performed molecular dynamics simulations on DNA segments with the consensus sequence for p53-specific binding, half site DNA-p53 complexes, and full site DNA-p53 complexes. We show that each DNA-bound p53 core domain caused a local DNA conformational change within the quarter site; upon the binding of the p53 dimer, there was an apparent DNA bending at the center of the half site; when bound with two p53 dimers, the full site DNAs with two different sequences bent 20 and 35 degrees, respectively. These results are in agreement with experimental observations. Our simulations demonstrate that the two p53 dimers favored a staggered conformation in which they make favorable interactions at the interface. This dimer-dimer interface organization necessitated conformational changes in the DNA, leading to the bending at the center of the full site, which in turn is dependent on the DNA sequence. Overall, our results provide the detailed atomic model for the DNA-p53 tetramer complex and delineate the roles of DNA-p53, p53 dimer-dimer interactions, and DNA sequence in specific p53 binding-induced DNA conformational changes.

Interactions between c-Jun, Nuclear Factor 1, and JC Virus Promoter Sequences: Implications for Viral Tropism

The infectious cycle of the human polyomavirus JC (JCV) is ultimately regulated in cellular nuclei at the level of viral protein expression and genomic replication. Such activity is prompted by interactions between variant nucleotide sequences within the JCV regulatory region (promoter) and cellular transcription factors that bind specific DNA consensus sites. In previous work we identified an NF-1 class member, NF-1X, as a critical transcription factor affecting the JCV cellular host range. Within variant JCV promoters, as well as other viral and cellular promoters, adjacently located NF-1 and AP-1 consensus sites are often found. The close proximity of these two binding sites suggests the opportunity for interaction between NF-1 and AP-1 proteins. Here, by electrophoretic mobility shift assays, we show temporal and dose-dependent interference by an AP-1 family member, c-Jun, upon NF-1 proteins binding an NF-1 consensus site derived from JCV promoter sequence. Moreover, as demonstrated by protein-protein interaction assays, we identify specific binding affinity independent of DNA binding between NF-1X and c-Jun. Finally, to compare the binding profiles of NF-1X and c-Jun on JCV promoter sequence in parallel with in vivo detection of viral activity levels, we developed an anchored transcriptional promoter (ATP) assay. With use of extracts from JCV-infected cells transfected to overexpress either NF-1X or c-Jun, ATP assays showed concurrent increases in NF-1X binding and viral protein expression. Conversely, increased c-Jun binding accompanied decreases in both NF-1X binding and viral protein expression. Therefore, inhibition of NF-1X binding by c-Jun appears to play a role in regulating levels of JCV activity.

Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Galphaq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment.

C/EBPα Zippers with AP-1 but Not Maf Proteins, and C/EBPα:AP-1 Heterodimers Preferentially Bind a Hybrid cis Element.

The leucine zipper (LZ) is an amphipathic α-helical motif that mediates protein:protein interactions via its hydrophobic surface. For bZIP transcription factors, dimerization mediated by the LZ positions the adjacent, N-terminal, basic region (BR) of each polypeptide to interact with the major groove of DNA. The C/EBPs are a sub-family of bZIP proteins that readily homo- or hetero-dimerize to bind C/EBP consensus DNA elements. The AP-1s are a second major bZIP subfamily in which c-Jun, JunB, or JunD heterodimerizes with Fos, FosB, Fra1, or Fra2 to bind an AP-1 site. In addition, c-Maf or MafB heterodimerize with AP-1s to bind related cis elements. C/EBPα, AP-1s, and Mafs have each been implicated as regulators of myeloid development. We have therefore assessed their ability to directly zipper and to alter each others DNA-binding specificities. Earlier work had found that exogenously expressed C/EBPα or C/EBPβ interact with c-Jun, but not if the LZ domain of either protein was deleted. The classic approach for demonstrating direct zippering between proteins which share the same DNA consensus site has been to use a gel shift assay. Interactions that did not involve zippering would not position the respective BRs to enable DNA contact. We have employed a related, novel assay to assess zippering between proteins with different binding specificities. The LZs of c-Jun, JunB, c-Fos, c-Maf, or MafB were swapped into full-length C/EBPα. These hybrid proteins were co-expressed with truncated proteins containing only the C/EBPα or C/EBPβ bZIP domains. Gel shift species of intermediate mobility were evident with the AP-1 but not the Maf LZs. As a second approach, we co-expressed c-Jun, JunB, or c-Fos with C/EBPα or with C/EBPαL12V in 293T cells. The L12V variant has two leucines in the LZ changed to valine and so cannot zipper. Each of the AP-1 proteins co-iped with C/EBPα but not with L12V. Although co-ip has not proven sensitive enough to detect endogenous zippering, even between C/EBPα and C/EBPβ, we obtained evidence for such interaction using confocal microscopy of U937 cells exposed to phorbol ester for 2 hrs to induce AP-1 protein expression. With C/EBPα labelled in the red, rhodamine channel and AP-1 proteins labelled individually in the green, FITC channel, co-localization of C/EBPα with either c-Jun or c-Fos, as indicated by yellow signal, was evident. Perhaps JunB was not expressed at sufficient levels to detect interaction even with this approach. As a tool for forcing hetero- or homo-dimerization between specfic bZIP proteins we have developed variants of the GCN4 LZ in which all of the salt bridge residues are either E (acidic) or K (basic). Neither C/EBPαLZE nor C/EBPαLKZ bind DNA but the combination does. We also generated cJunLZK and JunBLZK proteins and carried out gel shift assays using three oligos: JJ, a dyad symmetric AP-1 site; αα, a perfect C/EBP site; and αJ; a hybrid site. Whereas C/EBPαLZE:CEBPαLZK binds preferentially to the αα site, C/EBPαLZE:cJunLZK or C/EBPαLZE:JunBLZK bind preferentially to the hybrid site. Thus, zippering between C/EBPα and AP-1 proteins may redirect each other to novel cis elements and so novel genetic targets. We plan to employ these reagents to select additional binding sites in an unbiased assay and to probe the functional consequences of exogenous expression of C/EBP:AP-1 hybrid proteins. Results obtained may be relevant beyond myeloid development as these bZIP proteins regulate differentiation, proliferation, and survival in multiple lineages.

Smad3 Interacts with JunB and Cbfa1/Runx2 for Transforming Growth Factor-β1-stimulated Collagenase-3 Expression in Human Breast Cancer Cells

We have previously shown that transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.