Transcription regulation of development

We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBFmediated transcription. In the present study, we have examined the mechanism by which Rb represses UBFdependent rDNA transcription and determined if other Rb-like proteins have similar eects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb. Oncogene (2000) 19, 4988 ± 4999.

RESUMO -Aspectos fisiológicos e bioquímicos durante a germinação de sementes e crescimento inicial de plântulas de açaí foram investigados. Sementes coletadas de plantas crescendo na várzea e na terra firme foram utilizadas para determinar a influência das condições de normoxia (aeróbica) e de anoxia (anaeróbica) na germinação e no tempo inicial e médio do desenvolvimento da raiz e parte aérea. Após 75 dias, plantas germinadas sob normoxia foram transferidas para condições de alagamento. A quantificação das reservas da semente (lipídios, proteínas, açúcares solúveis e amido) foi monitorada em sementes quiescentes e germinadas em condições de normoxia e anóxicas, bem como após 5, 10 e 20 dias de crescimento das plantas. A atividade da enzima desidrogenase alcoólica (ADH) foi quantificada em raízes e folhas de plantas submetidas ao alagamento (parcial ou total) ou sem alagamento. As sementes não germinaram em condições de anoxia. Nas sementes, diferentes estratégias de mobilização de reservas (lipídios, proteínas, açúcares solúveis e amido) foram observadas, nas condições estudadas. A atividade da ADH foi induzida em condições de anoxia, sendo maior a atividade observada nas folhas. Este estudo demonstrou que, sob condições aeróbicas, o melhor desempenho das sementes de açaí, da várzea ou em terra firme, durante a germinação foi associado à mobilização de reservas e à tolerância das plântulas ao alagamento com o aumento da atividade da ADH. Concluiu-se que a palmeira açaí é a planta bem adaptada às condições de anoxia provocadas pelo alagamento.

G-quadruplexes (G4) are secondary four-stranded DNA helical structures consisting of guanine-rich nucleic acids, which can be formed in the promoter regions of several genes under proper conditions. Several cancer cells have been shown to emerge from genomic changes in the expression of crucial growth-regulating genes that allow cells to develop and begin to propagate in an undifferentiated state. Recent attempts have focused on producing treatments targeted at particular protein products of genes that are abnormally expressed. Many of the proteins found are hard to target and considered undruggable due to structural challenges, protein overexpression, or mutations that affect treatment resistance. The utilization of small molecules that stabilize secondary DNA structures existing in several possible oncogenes' promoters and modulate their transcription is a new strategy that avoids some of these problems. In this review, we outline the function of G-quadruplex stabilization in cancer by small-molecules with the aim to improve cancer therapy.

The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8 + T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues but it only accumulates in bone and calcified cartilage under physiological conditions. Available evidence indicates that it acts as a physiological inhibitor of mineralization. Runx2 is a transcription factor essential for bone formation in mammals, affecting osteoblast and chondrocyte differentiation by regulating key genes crucial for bone and cartilage development. Being an important cartilage-associated gene, MGP is a potential target for Runx2, and thus we have investigated the possible functional interactions between them. In A6 cells, Runx2 was found to modulate MGP transcription and deletion analysis of MGP distal and proximal promoter-luciferase constructs identified cis-regulatory regions. Interestingly, we have also identified a runx2-binding site that mediates transcriptional repression of XlMGP. Mutation of this element, located between -54 and + 33 bp, results in 18-fold up-regulation of transcription. Furthermore, and in addition to the previously reported Xlrunx2 types I and II, we have identified three transcripts encoding novel, truncated Xlrunx2 isoforms. Although only type I and type II could transactivate XlMGP, the truncated isoforms identified in this study, which result from alternative splicing, could be involved in negative regulation of MGP expression, as described for other RUNX2 truncated isoforms acting in other target genes. In vivo microinjection of XlMGP promoter constructs and runx2 mRNA confirmed that those promoters are targets for this transcription factor. These data also indicate that MGP is under dual regulation by runx2 through the use of various isoforms and context-dependent formation of transcriptional complexes.

Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3' untranslated region (UTR) of the RNA in mediating transport. Because the 3' UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3' UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3' UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among ...

Lithium, a well-known inhibitor of glycogen synthase kinase-3b (GSK3b), can improve bone formation by activating the Wnt/b-catenin signalling pathway. However, most studies have used higher doses of lithium, which potentially have adverse effects. Herein, we report that low dose lithium supplementation (10 mg/kg/d for 6 weeks) in mice results in a serum lithium concentration of 0.02 mM significantly inhibiting GSK3b while activating Wnt/b-catenin in bone. In turn, we observed a significant increase in the expression of osteoprotegerin (OPG), with unaltered expression of nuclear-factor kb ligand (RANKL), ultimately leading to a significant increase in the OPG/RANKL ratio. Altogether, our findings provide initial evidence that low dose lithium supplementation can promote the signalling pathways associated with bone formation.

Non-canonical, four-stranded nucleic acids secondary structures are present within regulatory regions in the human genome and transcriptome. To date, these quadruplex structures include both DNA and RNA G-quadruplexes, formed in guanine-rich sequences, and i-Motifs, found in cytosine-rich sequences, as their counterparts. Quadruplexes have been extensively associated with cancer, playing an important role in telomere maintenance and control of genetic expression of several oncogenes and tumor suppressors. Therefore, quadruplex structures are considered attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this review, we provide a general overview about recent research on the implications of quadruplex structures in cancer, firstly gathering together DNA G-quadruplexes, RNA G-quadruplexes as well as DNA i-Motifs.

Genes for -like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1, SIG2, and SIG3, for polypeptides possessing amino acid sequences for domains conserved in 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green f luorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may inf luence transcription by regulating RNA polymerase activity in a green tissue-specific manner. Plant Materials. A. thaliana ecotype Columbia was grown on vermiculite for 4 weeks at 22°C under 16-hr light͞8-hr dark. A. thaliana was also grown on a Murashige-Skoog (MS) agar medium (19) without sugar at 22°C in continuous light at 3,000

Human T-cell Lymphotropic Viruses type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma. Although associated with lymphocytosis, HTLV-2 infection is not associated with any malignant hematological disease. Similarly, no infection-related symptom has been detected in HTLV-3-infected individuals studied so far. Differences in individual Tax transcriptional activity might account for these distinct physiopathological outcomes. Tax-1 and Tax-3 possess a PDZ binding motif in their sequence. Interestingly, this motif, which is critical for Tax-1 transforming activity, is absent from Tax-2. We used the DNA microarray technology to analyze and compare the global gene expression profiles of different T- and non T-cell types expressing Tax-1, Tax-2 or Tax-3 viral transactivators. In a T-cell line, this analysis allowed us to identify 48 genes whose expression is commonly affected by all Tax proteins and are hence characteristic of the HTLV infection, independently of the ...

This review addresses a variety of mechanisms by which DNA repair interacts with transcription and vice versa. Blocking of transcriptional elongation is the best studied of these mechanisms. Transcription recovery after damage therefore has often been used as a surrogate marker of DNA repair in cells. However, it has become evident that relationships between DNA damage, repair, and transcription are more complex due to various indirect effects of DNA damage on gene transcription. These include inhibition of transcription by DNA repair intermediates as well as regulation of transcription and of the epigenetic status of the genes by DNA repair-related mechanisms. In addition, since transcription is emerging as an important endogenous source of DNA damage in cells, we briefly summarise recent advances in understanding the nature of co-transcriptionally induced DNA damage and the DNA repair pathways involved.

Potato cDNA microarray slides (TIGR 1 K) were used to compare changes in gene expression of two potato cell populations subjected to shock or gradual exposure to PEG-mediated water stress. A total of 180 transcripts were found to be statistically up-or down-regulated in PEG-shocked or gradually stressed cells, compared to untreated control cells. Up-regulated genes in shocked cells were prevalently involved in carbohydrate metabolism, cellular communication, and signal transduction. In cells stressed gradually, the most represented induced genes were those involved in signal transduction, in response to environmental signals and in the regulation of transcription and translation machineries, including the heat shock protein 90-2, ribosomal and RNA-binding proteins. The expression of selected up-regulated genes was also tested in potato plants subjected to slow soildrying conditions. Interestingly, the rgga gene, encoding an RNA-binding protein, was strongly induced in both leaves and roots of water-stressed potato plants, supporting its putative function in adaptive mechanisms to water stress. Pointing to differences in gene expression between shock-like and adaptive response, our findings might contribute to the controversial debate on the biological function of stress-responsive genes in cell damage repair or in restoring homeostasis and elaborating longer term responses for adaptation.

In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of P LAC , P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of P LAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.

Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription. We also found that factor occupancy generally correlates with high gene expression but that the loss of menin does not result in significant changes in most transcript levels. One exception is the developmentally programmed transcription factor, HLXB9, which is overexpressed in islets in the absence of menin. Our findings expand the realm of menin-targeted genes several hundred-fold beyond that previously described and provide potential insights to the endocrine tumor bias observed in MEN1 patients.

We studied the regulation of murine CD80, a gene whose basal transcriptional status was characterized by the presence of a stalled RNA polymerase II complex on the promoter-proximal region. Stimulus-induced activation of productive elongation involved a complex interplay of regulated events that included a synergy between ordered cofactor recruitment. This cascade of recruitments was initiated through the engagement of transcription factor NF-B, leading to the temporal association of histone acetyltransferases and the consequent selective acetylation of a transcription start site downstream nucleosome. This in turn culminated into the nucleosomal association of Brd4associated P-TEFb, a protein complex containing kinase specific for serine 2 of Rbp 1, the largest subunit of the carboxyl-terminal domain of RNA polymerase II. The consequent phosphorylation of serine 2 residues in CTD by CDK9 in the P-TEFb complex then facilitated escape of polymerase II into the productive elongation phase. Thus, the cooperative mechanisms that integrate between independent pathways characterize regulation of the elongation step of transcription, thereby providing another level at which specificity of gene regulation can be achieved.

HIV-1 gene expression requires both viral and cellular factors to control and coordinate transcription. While the viral factor Tat is known for its transcriptional transactivator properties, we present evidence for an unexpected function of Tat in viral splicing regulation. We used a series of HIV-1 reporter minigenes to demonstrate that Tat's role in splicing is dependent on the cellular co-transcriptional splicing activators Tat-SF1 and CA150. Surprisingly, we show that this Tat-mediated splicing function is independent from transcriptional activation. In the context of the full-length viral genome, this mechanism promotes an autoregulatory feedback that decreases expression of tat and favors expression of the env-specific mRNA. Our data demonstrate that Tat-mediated regulation of transcription and splicing can be uncoupled and suggest a mechanism for the involvement of specific transcriptional activators in splicing.

Background: Expression of signal transducer and activator of transcription 1 (STAT1), the mediator of interferon (IFN) signalling, is raised in synovial tissue (ST) from patients with rheumatoid arthritis (RA). Objectives: To determine the extent to which this pathway is activated by phosphorylation in RA synovium. Additionally, to investigate the cellular basis of STAT1 activation in RA ST. Methods: ST specimens from 12 patients with RA and 14 disease controls (patients with osteoarthritis and reactive arthritis) were analysed by immunohistochemistry, using antibodies to STAT1, tyrosine phosphorylated STAT1, and serine phosphorylated STAT1. Lysates of cultured fibroblast-like synoviocytes stimulated with IFNb were analysed by western blotting. Phenotypic characterisation of cells expressing STAT1 in RA ST was performed by double immunolabelling for STAT1 and CD3, CD22, CD55, or CD68. Results: Raised levels of total STAT1 protein and both its activated tyrosine and serine phosphorylated forms were seen in RA synovium as compared with controls. STAT1 was predominantly abundant in T and B lymphocytes in focal inflammatory infiltrates and in fibroblast-like synoviocytes in the intimal lining layer. Raised levels of STAT1 are sustained in cultured RA compared with OA fibroblast-like synoviocytes, and STAT1 serine and tyrosine phosphorylation is rapidly induced upon stimulation with IFNb. Conclusion: These results demonstrate activation of the STAT1 pathway in RA synovium by raised STAT1 protein expression and concomitantly increased tyrosine (701) and serine (727) phosphorylation. High expression of STAT1 is intrinsic to RA fibroblast-like synoviocytes in the intimal lining layer, whereas activation of the pathway by phosphorylation is an active process.

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-depend...

Search for a promoter element by RNA polymerase from the extremely large DNA base sequence is thought to be the slowest and rate-determining for the regulation of transcription process. Few direct experiments we described here which have tried to follow the mechanistic implications of this promoter search. However, once the promoter is located, transcription complex, constituting mainly the RNA polymerase molecule and few transcription factors has to unidirectionally clear the promoter and elongate the RNA chain through a series of steps which altogether define the initiation of transcription process. Thus, it appears that the promoter sequence acts as a trap for RNA polymerase associated with a large binding constant, although to clear the promoter and to elongate the transcript such energy barrier has to be overcome. Topological state of the DNA, particularly in the neighbourhood of the promoter plays an important role in the energetics of the whole process.

All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development. We used a unique combination of tools to obtain regional global-local alignments of orthologous loci. This approach takes into account shuffling of regulatory regions that are likely to occur over evolutionary distances greater than those separating mammalian genomes. This approach revealed one order of magnitude more vertebrate conserved elements than was previously reported in over 2,000 genes, including a high number of genes found in the membrane and extracellular regions. Our analysis revealed that 72% of the elements identified have undergone shuffling. We tested the abilit...

Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular mechanisms of action: ( i ) relatively slow genomic effects, such as the regulation of transcription by cognate T receptors (TR) and E receptors (ER); and ( ii ) relatively rapid nongenomic effects, such as kinase activation and calcium release initiated at the membrane by putative membrane receptors. Genomic and nongenomic effects were thought to be disparate and independent. However, in a previous study using a two-pulse paradigm in neuroblastoma cells, we showed that E acting at the membrane could potentiate transcription from an E-driven reporter gene in the nucleus. Because both T and E can have important effects on mood and cognition, it is possible that the two hormones can act synergistically. In this study, we demonstrate that early actions of T via TRα1 and TRβ1 can potentiate E-mediated transcription (genomic effects) from a consensus E response element (ERE)-driven reporter ge...

Background: Alzheimer's disease (AD) pathology shows characteristic 'plaques' rich in amyloid beta (Aβ) peptide deposits. Inflammatory process-related proteins such as pro-inflammatory cytokines have been detected in AD brain suggesting that an inflammatory immune reaction also plays a role in the pathogenesis of AD. Glial cells in culture respond to LPS and Aβ stimuli by upregulating the expression of cytokines TNF-α, IL-1β, and IL-6, and also the expression of proinflammatory genes iNOS and COX-2. We have earlier reported that LPS/Aβ stimulationinduced ceramide and ROS generation leads to iNOS expression and nitric oxide production in glial cells. The present study was undertaken to investigate the neuroprotective function of AICAR (a potent activator of AMP-activated protein kinase) in blocking the pro-oxidant/proinflammatory responses induced in primary glial cultures treated with LPS and Aβ peptide. To test the anti-inflammatory/anti-oxidant functions of AICAR, we tested its inhibitory potential in blocking the expression of pro-inflammatory cytokines and iNOS, expression of COX-2, generation of ROS, and associated signaling following treatment of glial cells with LPS and Aβ peptide. We also investigated the neuroprotective effects of AICAR against the effects of cytokines and inflammatory mediators (released by the glia), in blocking neurite outgrowth inhibition, and in nerve growth factor-(NGF) induced neurite extension by PC-12 cells. Results: AICAR blocked LPS/Aβ-induced inflammatory processes by blocking the expression of proinflammatory cytokine, iNOS, COX-2 and MnSOD genes, and by inhibition of ROS generation and depletion of glutathione in astroglial cells. AICAR also inhibited down-stream signaling leading to the regulation of transcriptional factors such as NFκB and C/EBP which are critical for the expression of iNOS, COX-2, MnSOD and cytokines (TNF-α/IL-1β and IL-6). AICAR promoted NGF-induced neurite growth and reduced neurite outgrowth inhibition in PC-12 cells treated with astroglial conditioned medium. The observed anti-inflammatory/anti-oxidant and neuroprotective functions of AICAR suggest it as a viable candidate for use in treatment of Alzheimer's disease.

Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription.

Objective. CD4؉ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T 505 in the protein kinase C␦ (PKC␦) activation loop. However, the mechanisms that prevent PKC␦ T 505 phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKC␦, contributing to impaired ERK pathway signaling in lupus T cells. Methods. CD4؉ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. Results. Treating CD4؉ T cells with peroxynitrite nitrated PKC␦, preventing PKC␦ T 505 phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKC␦ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKC␦, but not unmodified PKC␦, was refractory to PMA-stimulated T 505 phosphorylation, similar to PKC␦ in peroxynitrite-treated cells. Conclusion. Oxidative stress causes PKC␦ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKC␦ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

Objective. CD4؉ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T 505 in the protein kinase C␦ (PKC␦) activation loop. However, the mechanisms that prevent PKC␦ T 505 phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKC␦, contributing to impaired ERK pathway signaling in lupus T cells. Methods. CD4؉ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. Results. Treating CD4؉ T cells with peroxynitrite nitrated PKC␦, preventing PKC␦ T 505 phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKC␦ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKC␦, but not unmodified PKC␦, was refractory to PMA-stimulated T 505 phosphorylation, similar to PKC␦ in peroxynitrite-treated cells. Conclusion. Oxidative stress causes PKC␦ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKC␦ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

Objective. CD4؉ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T 505 in the protein kinase C␦ (PKC␦) activation loop. However, the mechanisms that prevent PKC␦ T 505 phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKC␦, contributing to impaired ERK pathway signaling in lupus T cells. Methods. CD4؉ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. Results. Treating CD4؉ T cells with peroxynitrite nitrated PKC␦, preventing PKC␦ T 505 phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKC␦ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKC␦, but not unmodified PKC␦, was refractory to PMA-stimulated T 505 phosphorylation, similar to PKC␦ in peroxynitrite-treated cells. Conclusion. Oxidative stress causes PKC␦ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKC␦ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

More than 55 million people worldwide are affected by Alzheimer's disease each year, and by 2050, cases could exceed 152 million. Current literature suggests that epigenetic dysregulation in gene expression is one key mechanism of neurodegeneration and pathogenesis of Alzheimer's disease (AD) (Ross, 2022). However, information needs to be more comprehensive regarding the impact of epigenetic factors on synaptic dysregulation and the lack of reproducibility of results experimentally. The research study aimed to examine β-amyloid and Tau activity from epigenetic factor Suv39h1 as a therapeutic target in drug discovery. Suv39h1 was chosen as a potential target for research as its function in creating gradients for histone methylation on histone 3 (H3) due to the similar function of Euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and EHMT2, shown to inhibit Amyloid-beta aggregation in AD. The study included a three-step data collection methodology, processing and modeling, and toxicity prediction. Data collection involved referencing functional relationships to identify similar inhibition patterns of Amyloid and Tau proteins with a review of Apolipoprotein E (ApoE) and EHMT1 increasing synaptic activity. Sequence alignment was also done during this phase to locate functional relationships using BLAST and STRING. Protein modeling and experimentation were done with ligandreceptor binding applications to determine efficacy. Lastly, toxicity prediction through SVM classification indicates drug development feasibility. The results indicated Suv39h1 as a viable factor associated with Tau aggregation. Although current literature has not yet identified the gene as an epigenetic factor involved in AD, the binding affinity results using Global Range Molecular Matching (GRAMM) demonstrate high efficacy. Overall, the results from the study indicate Suv39h1 as a functioning potential therapeutic target in AD, prompting further in-vitro and in-vivo research to confirm associations.

Multiple endocrine neoplasia type 1 (MEN1) is characterized by the occurrence of tumors of the parathyroids, neuroendocrine cells of the gastro-entero-pancreatic tract, and the anterior pituitary, including more than 20 other endocrine and nonendocrine tumor combinations. MEN1 gene, a tumor suppressor gene, encodes a protein, menin. Loss of heterozygosity (LOH) at 11q13 has been found in MEN1 tumors, but also in sporadic pituitary tumors and in 30-70% of sporadic parathyroids and endocrine pancreas tumors, a subset of those with LOH at 11q13 exhibiting mutations in the MEN1 gene. MEN1 mRNA and protein are expressed in all normal studied tissues, unexplaining the endocrine predominance of neoplasms. Mutations in the MEN1 gene frequently predict protein truncation possibly leading to inactivated function consistent with the idea of a tumor suppressor gene. Menin interacts with AP-1 transcription factor JunD, Smad3 and inhibits NFkB-mediated transactivation. Moreover, menin also interacts with pem, nm23, GFAP, the 32-kDa subunit of REPA, and nonmuscle myosin II-A heavy chain. Menin uncouples Elk-1, JunD, and c-Jun phosphorylation from MAPK activation. The effects of MEN1 in human neuroendocrine cells and its role in gene regulation remain still elusive. We studied the role of MEN1 mRNA and menin protein in the global gene expression network in skin fibroblasts from three MEN1 patients with heterozygote truncating mutation of gene and from healthy individuals: two sisters with a frame-shift, 738del4 (exon 3), introducing a premature stop codon, and a MEN1 individual with a nonsense mutation, R460X (exon 10). Densitometric analysis revealed a reduction of MEN1 mRNA expression in fibroblasts from patients vs healthy donor as also confirmed by Real Time RT-PCR. Contrarily, menin protein seems to be similarly expressed in the three MEN1 patients as also in the healthy control. These preliminary data show a discrepancy between menin-mRNA and menin protein expression: reduced menin-mRNA expression does not fit with the observed equal expression of menin protein. A mechanism of compensation at mRNA and/or protein levels may explain our results. The down-regulation of the wild-type allele by siRNA and ribozymes in heterozygous MEN1 mutants should give more informations about this hypothetical mechanism.

Endocrine pancreatic tumors are rare neoplasms that occur sporadically or as part of a multiple endocrine neoplasia type 1 (MEN1) syndrome. Germ-line mutations of the MEN1 gene, located at 11q13, have been demonstrated in MEN1 kindreds, and loss of heterozygosity (LOH) on 11q13 together with somatic MEN1 mutations have been detected in 20% of nonfamilial parathyroid tumors. Here, we examine 11 non-MEN1 malignant tumors of the endocrine pancreas, 9 nonfunctioning tumors, and 2 glucagonomas. LOH of at least one informative locus on 11q13 was found in 70% of the tumors. Three tumors displayed somatic mutations of the MEN1 gene together with LOH on 11q13, whereas the corresponding germ-line DNA was normal. These findings support the hypothesis that MEN1 gene mutations contribute to the tumorigenesis of nonfamilial, malignant endocrine pancreatic tumors.

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro...

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human OPEN ACCESS prostacyclin synthase (3.3 μM) with an EC 50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO 2 -radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

A quantitative structure activity relationship (QSAR) study on tetrasubstituted pyrazoles as high affinity ligands for the estrogen receptor (both ERα and ERβ subtypes) have been performed using various combinations of hydrophobic (MlogP), steric (MR) and electronic (Xeq) descriptors. The regression analysis of the data has shown better results in multiparametric regressions upon introduction of dummy parameters (indicator variables). The results suggest that the binding affinity for ERα and ERβ subtypes in tetrasubstituted pyrazoles is largely enhanced by the negative coefficient of MlogP and positive coefficient of MR descriptors.

Using the c-fos enhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fos promoter mutated at the C/EBP binding site upstream of luciferase, c-fos promoter activity is stimulated by GH 6-7-fold; wild type c-fos promoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2-5 min) increases binding of C/EBP␤ and C/EBP␦, to the c-fos C/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBP␤, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fos expression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBP␦ elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBP␤ and-␦ and increases levels of C/EBP␦. Although C/EBP␤ is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBP␤ and-␦ in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBP␤ and-␦ to the c-fos promoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBP␤ and-␦ to regulate GH-stimulated expression of c-fos.

In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of P LAC , P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of P LAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.

The process of spore formation in Bacillus subtilis is dependent upon a sophisticated program of gene expression that is regulated both temporally and spatially by a series of alternate sigma factors, in conjunction with a number of transcriptional regulators. One of these, SpoVT, regulates forespore-specific r G-dependent transcription and is related at the amino acid level to the major stationary phase sentinel, AbrB, whose mode of DNA recognition appears to be non-classical. Here, we report that the C-terminal domain of SpoVT is crucial to its correct folding and function, and how the DNA-binding domain from AbrB cannot complement the closely homologous domain of SpoVT in vivo. We also establish the oligomeric state of SpoVT and its component domains. Finally, we demonstrate that the regulation of transcriptional control by SpoVT is unexpectedly more complicated than its counterpart, AbrB, and that the latent non-specific DNA-binding activity of the N-terminal domain of SpoVT is modulated by the C-terminal domain, which perhaps in combination with another unknown factor, confers specificity.

The ability of the IHF-like proteins of Acinetobacter junii and Proteus vulgaris to interact with the HP attP and p P ihf sites of V DNA was investigated. IHF from A. junii and P. vulgaris was found to bind the examined ihf sites in a way similar to IHF from Escherichia coli as shown by gel mobility shift DNA binding assays and footprinting analysis. The three IHF proteins bound to the p P ihf site that overlaps the 335 region of that promoter and in vitro repression of transcription by each IHF was observed. These results confirm that IHF-like proteins from Gram-negative bacteria can recognize the same specific DNA sequences and appear to be important in regulation of transcription.

Based on previous studies suggesting that fibrinogen (Fg) might be a potential target for peroxynitrite (PN) action in plasma, we investigated the effects of PN on structure and hemostatic function of Fg in vitro. Using fluorescence and spectrophotometric methods, we estimated that about 0.5, 2 and 8 tyrosine residues per molecule were nitrated following the reaction of Fg at concentration 5.88 μM with 10, 100 and 1000 μM PN, respectively. At the same molar ratios of Fg to PN, about 0.01, 0.19 and 0.34 of tyrosine residues per molecule were oxidized to dityrosine and the amount of carbonyl groups in Fg increased 1.3-, 2,3-and 3.6-fold when compared to control Fg. SDS-PAGE analysis of PN-modified Fg suggests that inter-and intramolecular dityrosine cross-links occur between Aα chains of Fg. Vulnerability of Fg subunits to oxidative/nitrative modifications induced by PN was different. Within the Fg molecule, mainly αC domains as well as D domains (contrary to E domain) undergo the majority of the modifications. Low extent of nitration and oxidation of Fg molecule (induced by 10 μM PN) did not affect its clotting activity and susceptibility to degradation by plasmin. Modification of Fg induced by higher PN concentrations decreased these properties.

The positive transcription elongation factor b (P‐TEFb) is a heterodimeric complex composed of cyclin‐dependent kinase 9 and its regulator cyclin T1/2. It stimulates transcription elongation by phosphorylation of serine 2 residues in the carboxy‐terminal domain of polymerase II. 7SK RNA and HEXIM proteins can antagonize transcriptional stimulation by sequestering P‐TEFb in a catalytically inactive ribonucleoprotein (RNP). Here, we show that the previously uncharacterized La‐related protein 7 (LARP7) has a role in 7SK‐mediated regulation of transcription. LARP7 binds to the highly conserved 3′‐terminal U‐rich stretch of 7SK RNA and is an integral part of the 7SK RNP. On stimulation, LARP7 remains associated with 7SK RNA, whereas P‐TEFb is released. Interestingly, reduction of LARP7 by RNA interference enhances transcription from cellular polymerase II promoters, as well as a TAT‐dependent HIV‐1 promoter. Thus, LARP7 is a negative transcriptional regulator of polymerase II genes, acti...

ABSTRACTDNA methylation contributes to the chromatin conformation that represses transcription of human papillomavirus type16 (HPV-16), which is prevalent in the etiology of cervical carcinoma. In an effort to clarify the role of this phenomenon in the regulation and carcinogenicity of HPV-16, 115 clinical samples were studied to establish the methylation patterns of the 19 CpG dinucleotides within the long control region and part of the L1 gene by bisulfite modification, PCR amplification, DNA cloning, and sequencing. We observed major heterogeneities between clones from different samples as well as between clones from individual samples. The methylation frequency of CpGs was measured at 14.5%. In addition, 0.21 and 0.23%, respectively, of the CpA and CpT sites, indicators of de novo methylation, were methylated. Methylation frequencies exceeded 30% in the CpGs overlapping with the L1 gene and were about 10% for most other positions. A CpG site located in the linker between two nuc...

Transcriptional enhancers are key determinants of developmentally regulated gene expression. Models of enhancer function must distinguish between analog or digital control of transcription, as well as their requirement to initiate or maintain transcriptional activity of a gene. In light of a recent study by Chong and colleagues (pp. 659–669) providing evidence of a transient requirement of an enhancer associated with the CD4 gene, we discuss possible mechanisms by which transcriptional memory can be propagated in the absence of enhancers.

Here, initial investigations on the interaction of benzo[b]fluorenone (BF) with the human telomeric forming oligonucleotide HT4 using circular dichroism (CD), thermal melting and steady-state fluorescence are reported. CD studies suggest that BF exhibits binding selectivity for G-quadruplex over model double-stranded DNA structures. BF binds to hybrid-mixed type quadruplex structures in the presence of physiological concentrations of K þ ions (100mM). However, even in the absence of salt, BF can induce an anti-parallel type quadruplex conformation. Thermal melting studies on quadruplex HT4 in the presence and absence of salt show BF has little effect on the melting temperature, suggesting that BF may associate with the G-quadruplex structure through a non-intercalating mode. As BF is known to be a solvatochromic fluorophore, steady state fluorescence emission spectra were taken for BF titrated with the HT4 quadruplex formed at 100mM K þ , to explore the ability of BF to act as a potential probe of the environment of its binding site. Based on the red shift of BF emission, BF/quadruplex association appears to involve a relatively polar and hydrogen-bonded environment.

B cells are essential to the immune response in health and disease. Results from knockout (KO) mice for different members of the nuclear factor-κB (NF-κB) family have highlighted the importance of this transcription factor in B cell development and function. The recent generation of additional KO mice for adapters and kinases implicated in NF-κB activation, including several protein kinase C isoforms, has provided new insights into the roles of these proteins in B cell signalling. These studies have also given rise to a number of important questions that must be answered with further experimentation to establish accurately the signalling pathways that regulate B-cell function through NF-κB.

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro...

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)–endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial–mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, an...

The process of post-translational covalent modifications of proteins represents a transcription-independent regulatory mechanism allowing rapid alteration of protein activity and function in response to various intra-and extracellular stimuli. Lysine methylation (KM) was deemed to be a constant covalent mark, providing long-term signaling, including the histone-dependent mechanism for transcriptonal memory. Only recently has it become apparent that lysine methylation, similar to other covalent modifications, is transient and can be dynamically regulated by an opposing activity, de-methylation. These discoveries accelerated a systematic search for other nonhistone substrates of lysine methylation, especially among transcription factors. Recent findings suggest that KM affects gene expression not only at the level of chromatin, but also by modifying transcription factors.