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T Maniatis

Publications and source records attributed to T Maniatis.

At least 73 records · Page 4Linked to original sources
PubMed1995

Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1.

Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene in endothelial cells is induced by lipopolysaccharide and the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA-protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3' of the TATA element. We have identified heterodimeric p65 and p50 as the NF-kappa B species binding to the VCAM1 promoter in TNF-alpha-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and high-mobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assay in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1.

Base Sequence↗
PubMed1995

Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers.

Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.

Base Sequence↗
PubMed1994

The high mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms.

The high mobility group protein HMG I(Y) stimulates the binding of a specific isoform of the activating transcription factor 2 (ATF-2(195)) to the interferon beta (IFN-beta) gene promoter. HMG I(Y) specifically interacts with the basic-leucine zipper region of ATF-2(195), and HMG I(Y) binds to two sites immediately flanking the ATF-2 binding site of the IFN-beta promoter. Here, we show that HMG I(Y) can stimulate the binding of ATF-2(195), at least in part, by promoting ATF-2 dimerization. In addition, we report the characterization of a naturally occurring isoform of ATF-2 (ATF-2(192)) that binds specifically to the IFN-beta promoter but is unable to interact with HMG I(Y). Remarkably, HMG I(Y) inhibits the binding of ATF-2(192) to the IFN-beta promoter. Thus, the ability of HMG I(Y) to specifically interact with ATF-2 correlates with its ability to stimulate ATF-2 binding to the IFN-beta promoter. Comparisons of the amino acid sequences of the basic-leucine zipper domains of ATF-2(195) and ATF-2(192) suggest that HMG I(Y) interacts with a short stretch of basic amino acids near the amino terminus of the basic-leucine zipper domain of ATF-2(195).

Activating Transcription Factor 2↗
PubMed1994

The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B.

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.

Adenosine Triphosphate↗
PubMed1994

An HMG-like protein that can switch a transcriptional activator to a repressor.

One protein can activate some genes and repress others in the same cell. The Drosophila protein Dorsal (which, like the human protein NF-kappa B3, is a member of the Rel family of transcriptional activators) activates the twist gene and represses the zen gene in the ventral region of early embryos. Here we describe a Drosophila HMG1 protein, called DSP1 (dorsal switch protein), that converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed a negative regulatory element (NRE), found adjacent to Dorsal-binding sites in the zen promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta (IFN-beta) enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription.

Amino Acid Sequence↗
PubMed1994

A splicing enhancer exhibits both constitutive and regulated activities.

The Drosophila proteins Transformer (Tra) and Transformer2 (Tra2) regulate the sex-specific alternative splicing of Drosophila doublesex (dsx) pre-mRNA by specifically binding to a splicing enhancer (dsx repeat element; dsxRE) located 300 nucleotides (nt) downstream from a female-specific 3' splice site. In this paper we show that the dsxRE can function as a Tra and Tra2-independent splicing enhancer in vitro when located within 150 nucleotides of the 3' splice site. Based on the relative levels of SR proteins that bind stably to the dsxRE in the presence or absence of Tra and Tra2, we propose that the constitutive splicing activity of the dsxRE is mediated by its weak interactions with SR proteins and possibly other general splicing factors. In contrast, Tra and Tra2 allow the dsxRE to function at a distance from the intron by stabilizing the interactions between these proteins and the dsxRE.

Alternative Splicing↗
PubMed1994

The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2.

We have investigated the function of different structural domains of the Drosophila splicing regulator Transformer 2 (Tra2). We find that the ribonucleoprotein consensus sequence (RNP-CS) of Tra2 is required for male fertility and positive and negative control of alternative splicing in transgenic flies, as well as for in vitro binding of recombinant Tra2 to doublesex and tra2 pre-mRNAs. Thus, all of the known functions of Tra2 require specific protein-RNA interactions. We also show that one of the two arginine-serine (RS)-rich domains of Tra2 is dispensable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Significantly, the essential RS domain is also required for specific protein-protein interactions. We find that Tra2 interacts with itself, with the splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demonstrate that both protein-RNA and protein-protein interactions are involved in tra2-dependent activation and repression of alternative splicing.

Animals↗
PubMed1994

A striking similarity in the organization of the E-selectin and beta interferon gene promoters.

Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters.

Activating Transcription Factor 2↗
PubMed1993

Specific interactions between proteins implicated in splice site selection and regulated alternative splicing.

Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. We report that the splicing factors SC35 and SF2/ASF specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing.

Alternative Splicing↗
PubMed1993

Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements.

The high mobility group protein HMG I(Y) and the transcription factor NF-kappa B are required for the activity of positive regulatory domain II (PRDII), a virus-inducible regulatory element of the human interferon-beta gene promoter. In this paper we provide evidence that HMG I(Y) is also required for the activity of PRDIV, a regulatory element that synergizes with PRDII. In this case, HMG I(Y) stimulates binding of activating transcription factor 2 (ATF-2) and the assembly of inducible complexes containing ATF-2 and c-Jun. Remarkably, HMG I(Y) also specifically interacts with the leucine zipper/basic region of ATF-2, and ATF-2 in turn interacts with NF-kappa B. We therefore propose that the HMG I(Y) plays a critical structural role in establishing transcriptional synergy between PRDII and PRDIV by promoting the activities and/or binding of NF-kappa B and ATF-2 and by facilitating their interaction.

Activating Transcription Factor 2↗
PubMed1993

A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA.

Female-specific splicing of Drosophila doublesex (dsx) pre-mRNA is regulated by the products of the transformer (tra) and transformer 2 (tra2) genes. In this paper we show that Tra and Tra2 act by recruiting general splicing factors to a regulatory element located downstream of a female-specific 3' splice site. Remarkably, Tra, Tra2, and members of the serine/arginine-rich (SR) family of general splicing factors are sufficient to commit dsx pre-mRNA to female-specific splicing, and individual SR proteins differ significantly in their ability to participate in commitment complex formation. Characterization of the proteins associated with affinity-purified complex formed on dsx pre-mRNA reveals the presence of Tra, Tra2, SR proteins, and additional unidentified components. We conclude that Tra, Tra2, and SR proteins are essential components of a splicing enhancer complex.

Animals↗
PubMed1993

A Drosophila GATA family member that binds to Adh regulatory sequences is expressed in the developing fat body.

We have identified a Drosophila transcription factor that binds a sequence element found in the larval promoters of all known alcohol dehydrogenase (Adh) genes. DNA sequence analysis of cDNA clones encoding this protein, box A-binding factor (ABF), reveals that it is a member of the GATA family of transcriptional regulatory factors. ABF-binding sites within the D. mulleri and D. melanogaster larval Adh promoters function as positive regulatory elements and in cotransfection experiments, ABF functions as a transcriptional activator. In further support of a role for ABF in the regulation of Adh expression, ABF mRNA is expressed in the embryonic fat body, a tissue that contains high levels of Adh mRNA. Our studies demonstrate that the fat body develops from segmentally repeated clusters of mesodermal cells, which later expand and coalesce to form the mature fat body. These observations establish ABF as the earliest known fat body precursor marker in the Drosophila embryo. Together with the established role of GATA factors during mammalian development, these results suggest that ABF may play a key role in the organogenesis of the fat body.

Alcohol Dehydrogenase↗
PubMed1993

A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster.

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknüllt.

Amino Acid Sequence↗
PubMed1992

Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells.

Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.

Actins↗
PubMed1992

General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro.

Animals↗
PubMed1992

The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene.

In this paper, we show that both NF-kappa B and the high mobility group protein I(Y) (HMG I(Y)) are required for virus induction of the human interferon-beta (IFN-beta) gene. NF-kappa B binds to the terminal regions of a 10 bp regulatory sequence through contacts in the major groove. while HMG I(Y) recognizes the central region of the same sequence through contacts in the minor groove. Mutations that interfere with binding of either protein decrease the level of virus induction, and activation of the gene can be blocked by either NF-kappa B or HMG I(Y) antisense RNA. HMG I(Y) stimulates the binding of NF-kappa B to the IFN-beta promoter, and it may also function as a promoter-specific accessory factor for NF-kappa B transcriptional activity.

Amino Acid Sequence↗