NF-Y Sites Research Articles

Overexpression and alternative splicing of NF-YA in breast cancer

NF-Y is a CCAAT-binding trimeric transcription factor, whose regulome, interactome and oncogenic potential point to direct involvement in cellular transformation. Yet little is known about the levels of NF-Y subunits in tumors. We focused on breast carcinomas, and analyzed RNA-Seq datasets of TCGA and 54 BRCA cell lines at gene and isoforms level. We partitioned all tumors in the four major subclasses. NF-YA, but not histone-fold subunits NF-YB/NF-YC, is globally overexpressed, correlating with the proliferative Ki67 marker and a common set of 840 genes, with cell-cycle, metabolism GO terms. Their promoters are enriched in NF-Y, GC-rich and E2F sites. Surprisingly, there is an isoform switch, with the “short” isoform -NF-YAs- becoming predominant in tumors. E2F genes are also overexpressed in BRCA, but no switch in isoforms is observed. In Basal-like Claudinlow cell lines and tumors, expression of NF-YAl -long- isoform is high, together with 11 typical EMT markers and low levels of basal Keratins. Analysis of Progression-Free-Intervals indicates that tumors with unbalance of NF-YA isoforms ratios have worst clinical outcomes. The data suggest that NF-YA overexpression increases CCAAT-dependent, pro-growth genes in BRCA. NF-YAs is associated with a proliferative signature, but high levels of NF-YAl signal loss of epithelial features, EMT and acquisition of a more aggressive behavior in a subset of Claudinlow Basal-like tumors.

Transcriptional regulation of the porcine miR-17-92 cluster.

The miR-17-92 cluster has been involved in the cell cycle, apoptosis, and signaling. However, its transcriptional regulation has not been fully characterized. To elucidate the transcriptional regulation, the promoter of miR-17-92 was analyzed in detail in pig here. We found that, as an intronic miRNA, porcine miR-17-92 cluster was regulated by two independent promoters, an A/T-rich region directly upstream of the miR-17-92 coding sequence, and a G/C-rich region corresponding to the host gene promoter of the human miR-17-92 cluster. Several cis-regulatory elements were identified including sites for c-Myc, NFY, E2F3, and SP1, among which NFY and c-Myc sites were present in both A/T- and G/C-rich regions, while E2F3 and SP1 sites only existed in G/C-rich region. Sites for c-Myc, E2F3, and SP1 were positive for regulating transcription. NFY sites played bipartite roles, functioning as a repressor for the A/T-rich region, and as an activator for the G/C-rich region. Additionally, we found that levels of individual miRNAs in the cluster were not promoted completely in parallel with each other or with pri-miR-17-92 by the A/T-rich region, through using a self-made vector by modifying pGL3-basic in which firefly luciferase gene was replaced with an miR-17-92 cluster and a direct upstream A/T-rich region. The expression regulation of miR-17-92 is complicated and the results will contribute to further revealing the regulatory mechanisms under the expression of the miR-17-92 cluster.

Regulation of Stearoyl-Coenzyme A Desaturase 1 by trans-10,cis-12 Conjugated Linoleic Acid via SREBP1 in Primary Goat Mammary Epithelial Cells

trans-10, cis-12 Conjugated linoleic acid (t10c12-CLA) is a biohydrogenation intermediate in the rumen that inhibits mammary fatty acid de novo synthesis in lactating dairy goats. However, the underlying molecular pathways in milk-lipid metabolism affected by t10c12-CLA are not completely understood. The present study investigated the lipid-regulation mechanisms in goat mammary epithelial cells (GMECs) in response to t10c12-CLA. Gene-expression analysis indicated sterol-regulatory-element-binding transcription factor1 ( SREBF1) and its putative target gene stearoyl-CoA desaturase ( SCD1) were down-regulated (fold changes of 0.33 ± 0.04, P < 0.05, and 0.19 ± 0.01, P < 0.01, respectively). Concentrations of cellular palmitoleic acid (C16:1) and oleic acid (C18:1) were decreased (1.12 ± 0.05 vs 1.69 ± 0.11% and 15.70 ± 0.44 vs 24.97 ± 0.82%, respectively, P < 0.01), whereas those of linoleic acid (C18:2) were increased (5.00 ± 0.14 vs 3.81 ± 0.25%, P < 0.05); the desaturation indices of C16 and C18 were decreased in response to t10c12-CLA treatment (6.90 ± 0.05 vs 8.00 ± 0.30% and 61.41 ± 0.65 vs 67.73 ± 1.33%, respectively, P < 0.05). A luciferase-activity assay indicated that deletion of the sterol-response-element (SRE) site and the nuclear-factor (NF-Y) site in the SCD1-promoter region (-511/+65 bp) suppressed the regulatory effect of t10c12-CLA. Overexpression of SREBF1 partly counteracted the inhibitory effect of t10c12-CLA on de novo fatty acid synthesis. Overall, t10c12-CLA causes an inhibition of fatty acid synthesis and desaturation and regulates SCD1 expression by affecting the binding of SREBP1 protein to the SRE and NF-Y sites.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.

Curcumin Downregulates Human GM3 Synthase (hST3Gal V) Gene Expression with Autophagy Induction in Human Colon Carcinoma HCT116 Cells.

Our recent report showed that curcumin, polyphenolic compound isolated from the herb Curcuma longa, upregulated the gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis with autophagy induction in human lung adenocarcinoma A549 cells. In this study, on the contrary to this finding, we demonstrated that curcumin downregulated the gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 synthesis with autophagy induction in human colon carcinoma HCT116 cells. To clarify the mechanism leading to the downregulation of hST3Gal V gene expression in curcumin-treated HCT116 cells, we analyzed the curcumin-inducible promoter of the hST3Gal V gene by luciferase reporter assays. Promoter deletion analysis demonstrated that the -177 to -83 region, which includes putative binding sites for transcription factors NFY, CREB/ATF, SP1, EGR3, and MZF1, acts as the curcumin-responsive promoter of the hST3Gal V gene. Site-directed mutagenesis and chromatin immunoprecipitation analysis demonstrated that the CREB/ATF binding site at -143 is pivotal for curcumin-induced downregulation of hST3Gal V gene in HCT116 cells. The transcriptional activation of hST3Gal V in HCT116 cells was significantly repressed by an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that AMPK signal pathway mediates hST3Gal V gene expression in HCT116 cells.

CREB, NF-Y and MEIS1 conserved binding sites are essential to balance Myostatin promoter/enhancer activity during early myogenesis.

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer (Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors.

Upregulation of Human ST8Sia VI (α2,8-Sialyltransferase) Gene Expression by Physcion in SK-N-BE(2)-C Human Neuroblastoma Cells.

In this research, we firstly demonstrated that physcion, an anthraquinone derivative, specifically increased the expression of the human α2,8-sialyltransferase (hST8Sia VI) gene in SK-N-BE(2)-C human neuroblastoma cells. To establish the mechanism responsible for the up-regulation of hST8Sia VI gene expression in physcion-treated SK-N-BE(2)-C cells, the putative promoter region of the hST8Sia VI gene was functionally characterized. Promoter analysis with serially truncated fragments of the 5′-flanking region showed that the region between −320 and −240 is crucial for physcion-induced transcription of hST8Sia VI in SK-N-BE(2)-C cells. Putative binding sites for transcription factors Pax-5 and NF-Y are located at this region. The Pax-5 binding site at −262 to −256 was essential for the expression of the hST8Sia VI gene by physcion in SK-N-BE(2)-C cells. Moreover, the transcription of hST8Sia VI induced by physcion in SK-N-BE(2)-C cells was inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, but not c-Jun N-terminal kinase (JNK) inhibitor SP600125. These results suggest that physcion upregulates hST8Sia VI gene expression via ERK and p38 MAPK pathways in SK-N-BE(2)-C cells.

ORF50-dependent and ORF50-independent activation of the ORF45 gene of Kaposi's sarcoma-associated herpesvirus

The ORF45 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a multifunctional tegument protein. Here, we characterize the transcriptional control of the ORF45 gene and show that its promoter can be activated by ORF50 protein, a latent-lytic switch transactivator. The ORF45 promoter can also be induced by sodium butyrate (SB), a histone deacetylase inhibitor, in the absence of ORF50 protein. Although SB induces the ORF45 gene independently of ORF50, its full activation may require the presence of ORF50. Deletion and point mutation analyses revealed that two RBP-Jκ-binding sites in the ORF45 promoter confer the ORF50 responsiveness, whereas NF-Y and Sp1-binding sites mediate the response to SB. Direct binding of NF-Y, Sp1, or RBP-Jκ protein to the ORF45 promoter is required for the promoter activation induced by SB or by ORF50. In conclusion, our study demonstrates both ORF50-dependent and ORF50-independent transcriptional mechanisms operated on the activation of the ORF45 gene.

NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000–15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.

The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

Totipotency of embryonic stem cells (ESCs) is controlled at the transcriptional level by a handful of transcription factors (TFs) that promote stemness and prevent differentiation. One of the most enriched DNA elements in promoters and enhancers of genes specifically active in ESCs is the CCAAT box, which is recognized by NF-Y, a trimer with histone-like subunits--NF-YB/NF--YC--and the sequence-specific NF-YA. We show that the levels of the short NF-YA isoform--NF-YAs--is high in mouse ESCs (mESCs) and drops after differentiation; a dominant negative mutant affects expression of important stem cells genes, directly and indirectly. Protein transfections of TAT-NF-YAs stimulate growth and compensate for withdrawal of leukemia inhibitory factor (LIF) in cell cultures. Bioinformatic analysis identifies NF-Y sites as highly enriched in genomic loci of stem TFs in ESCs. Specifically, 30%-50% of NANOG peaks have NF-Y sites and indeed NF-Y-binding is required for NANOG association to DNA. These data indicate that NF-Y belongs to the restricted circle of TFs that govern mESCs, and, specifically, that NF-YAs is the active isoform in these cells.

Characterization of molecular clones of porcine endogenous retrovirus-A containing different numbers of U3 repeat boxes in the long terminal repeat region

When full-length molecular clones of porcine endogenous retrovirus (PERV)-A and porcine endogenous retrovirus (PERV)-B were passaged on human cells, an increase in the length of the long terminal repeat (LTR) was reported. A 39-bp repeat box in the LTR U3 region was multimerized dynamically upon replication, acting as a viral enhancer element that contains binding sites for nuclear transcription factor NF-Y. To analyze the optimum number of 39-bp repeats for viral replication, molecular clones of PERV-A with one, two, three, and four 39-bp repeats were constructed. Each full-length PERV-A molecular clone contained a different number of 39-bp repeat boxes and was used to transfect human 293 cells, the relative transcriptional activity of the LTRs 48 h posttransfection was determined. PERV LTRs containing 3 copies of a 39-bp repeat showed the strongest promoter activity by real-time reverse transcription PCR in human 293 cell lines. Virions generated by the transfection of a provirus with 3 enhancer repeats replicated efficiently in human cells and 2.5×10(4)virion copies/μL were released. Although the transcriptional activity of all PERV-A LTRs was lower than 293 cells (293-PERV-PK-CIRCE) infected productively with PERV-A and PERV-B. It was found that 3 was the optimal number of 39-bp repeats for viral replication. This molecular clone with a higher replication capacity could be used to study the biology of porcine endogenous retrovirus by genetic approaches or in vivo infection experiments.

Basal transcription of the human TBX3 gene, a key developmental regulator which is overexpressed in several cancers, requires functional NF-Y and Sp1 sites

TBX3 is a member of the T-box family of genes that encode developmentally important transcription factors. Mutations resulting in decreased levels of functional TBX3 lead to Ulnar-Mammary Syndrome and increased levels of TBX3 have been linked to several cancers. To understand the mechanisms regulating TBX3 expression we have previously cloned the 5′-flanking region of the human TBX3 gene and here we describe cis-elements required for its basal transcription. Using site-directed mutagenesis, luciferase reporter assays and in vitro and in vivo DNA binding experiments we identify a Sp1 element and two CCAAT boxes to be essential for basal TBX3 promoter activity. Our results are consistent with reports that these sites are necessary for efficient basal transcription in genes which lack TATA boxes or an Initiator which we show to be the case for TBX3.

Genome wide study of NF-Y type CCAAT boxes in unidirectional and bidirectional promoters in human and mouse

A subset of CCAAT boxes is known as binding sites for the transcription factor NF-Y. We characterize their number, mismatches to the consensus sequence, and locations in bidirectional and unidirectional promoter sequences in human and mouse. We confront the findings with an analytical null model of DNA sequences and find that NF-Y type CCAAT boxes play key, but distinct roles in the two types of promoters. They are found above chance in both, but in unidirectional only when having few mismatches. In bidirectional, the relative positions of multiple boxes differ from what is expected by chance, suggesting the need for contiguity. In agreement, when there are four boxes (four-box configurations), these have much lower number of mismatches than expected in bidirectional promoters alone. Positioning of the first box differs in the two types of promoters and the null model, and mismatches and positioning are found to be correlated. Finally, four-box configurations are conserved between human and mouse, supporting the relevance of the findings. We conclude that bidirectional and unidirectional promoters, while sharing some similarities, appear to possess distinct regulatory mechanisms at the sequence level.

NF-Y joins E2Fs, p53 and other stress transcription factors at the apoptosis table

NF-Y joins E2Fs, p53 and other stress transcription factors at the apoptosis table

Abstract 4990: Nuclear Factor-Y (NF-Y) regulates the transcription of the human aldo-keto reductase 1C1 (AKR1C1) gene*

Abstract Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Here, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene. The 5′ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5′-flanking, ∼3.0 kb region of the human AKR1C1 gene, identified the region between −128 to −88 as the minimal proximal promoter that is essential for basal transcription of AKR1C1 in human ovarian (2008 &amp; 2008/C13*), lung (H23 &amp; A549) and liver carcinoma (HepG2) cells. Based on computational analysis, a set of potential putative transcription factor binding sites for Oct1, Sp1, NF-Y, C/EBP, p40X, USF, NF1 and AP-2 were identified in this region. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were essential for controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at −109ATTGG−105 of the AKR1C1 gene. These results indicate that the NF-Y transcription factor regulates the basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4990.

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Transcriptional effects of Colony-stimulating factor-1 in mouse macrophages

Colony-stimulating factor-1 (CSF-1) is a major regulator of macrophage development. CSF-1-dependent signalling has been implicated in proliferation, survival, and differentiation of mononuclear phagocytes, however, relatively little is known about the effects of CSF-1 on macrophage gene transcription and on CSF-1-responsive gene promoters. We used a combination of transcription profiling and in silico motif search to characterize genes that are regulated in mature bone marrow-derived macrophages cultured in the presence or absence of CSF-1. The expression of many known differentiation-associated macrophage markers was not significantly affected in the absence of CSF-1. Genes repressed by CSF-1 comprised a considerable number of granulocyte-specific genes. The respective gene promoters; however, were not significantly enriched for specific DNA patterns, suggesting that these genes are regulated by promoter-distal elements or at a post-transcriptional level. Genes downregulated upon CSF-1 deprivation showed a highly significant association with cell division which is in line with the known role of CSF-1 as a proliferation stimulus for mouse macrophages. Interestingly, three DNA patterns were significantly co-enriched in CSF-1-dependent gene promoters, including motifs related to NFY, CHR, and E2F sites. These motifs showed a strong positional preference on target promoters at −60, −30 and 0 bp upstream of the transcription start site, and define the common promoter structure of CSF-1-responsive genes.

N-3 polyunsaturated fatty acids suppress insulin-induced SREBP-1c transcription via reduced trans-activating capacity of LXRα

Insulin coordinately up-regulates lipogenic gene transcription via induction of sterol regulatory element binding protein-1c (SREBP-1c). Conversely, polyunsaturated fatty acids (PUFA) decrease lipogenic gene transcription via suppression of SREBP-1c. We therefore examined the ability of n-3 PUFA to mitigate induction of SREBP-1c and its downstream lipogenic targets by insulin in primary rat hepatocyte cultures. Insulin induced expression of SREBP-1c mRNA 5-6 fold as well as rat SREBP-1c promoter activity. These effects were prevented by the n-3 fatty acids eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3, DHA), but not by the monounsaturated fatty acid oleic acid (18:1 n-6, OLA). N-3 fatty acids also effectively prevented insulin induction of the downstream lipogenic enzyme targets fatty acid synthase (FAS) and acetyl carboxyl coenzyme acetyltransferase-1 (ACC-1), and reduced de novo lipogenesis. The SREBP-1c promoter contains an insulin response unit consisting of tandem LXRα response elements (LXREs) as well as sites for NF-Y, Sp1, and SREBP-1c itself. The LXREs were identified as a primary site mediating suppression of SREBP-1c transcription by n-3 PUFA. DHA effectively prevented LXRα-dependent activation of both the wild type SREBP-1c promoter and the synthetic LXRE-driven promoter, and significantly blunted LXRα-dependent activation of a Gal4-LXRα chimeric protein thus demonstrating that n-3 PUFA effectively mitigate induction of SREBP-1c by insulin via reduced trans-activation of LXRα.

The Induction of IL-10 by Zymosan in Dendritic Cells Depends on CREB Activation by the Coactivators CREB-Binding Protein and TORC2 and Autocrine PGE2

Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated kappaB-binding activity, but inhibition of NF-kappaB was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE(2), and inhibitors of PKA, COX, and glycogen-synthase kinase-3beta showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE(2) and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3beta, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.

Long-chain polyunsaturated fatty acid synthesis in fish: Comparative analysis of Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.) Δ6 fatty acyl desaturase gene promoters

Fish vary in the ability to biosynthesise n-3 long-chain polyunsaturated fatty acids (LC-PUFA), with marine fish such as cod being inefficient in comparison to freshwater and salmonid fish. We investigated differences in the gene promoters of Delta6 fatty acyl desaturase (Delta6 FAD), a critical enzyme in LC-PUFA biosynthesis, in cod and salmon. Progressive deletions and targeted mutations of the promoters were tested for activity in a transfected Atlantic salmon (AS) cell line under low or high LC-PUFA treatment, and regions sufficient to direct transcription were identified. Comparison of these regions with sequences of corresponding regions of Delta6 FAD genes from mammals, amphibians and fish indicated a remarkable conservation of binding sites for SREBPs and NF-Y. In addition to these sites, a site was identified in salmon with similarity to that recognised by Sp1 transcription factor, and which was required for full expression of the salmon Delta6 FAD gene. The cod promoter was less active and lacked the Sp1 site. Eicosapentaenoic acid suppressed LC-PUFA synthesis in AS cells and also suppressed activity of the salmon Delta6 FAD promoter although this activity was likely mediated through sites other than Sp1, possibly similar to those recognised by NF-Y and SREBP transcription factors.