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Two tightly-linked Drosophila male accessory gland transcripts with the same developmental expression derive from independent transcription units.

Acp26Aa and Acp26Ab are Drosophila male accessory gland transcripts that are tightly linked and transcribed from the same DNA strand. Despite their being separated by 20 base pairs, the transcripts show identical responses to several developmental signals. These observations make it important to determine whether the 26A region contains two separable genes with the same developmental expression or a single developmentally regulated transcription unit whose product is processed to yield Acp26Aa and Acp26Ab. We show that Acp26Aa and Acp26Ab are separate mRNAs using a reverse transcription-polymerase chain reaction assay and reporter gene fusions. We also show that the regulatory elements for Acp26Ab lie within a fragment containing the intergenic region and transcribed sequences of Acp26Aa and Acp26Ab.

Animals↗

Transcriptional hierarchy in Xenopus embryogenesis: HNF4 a maternal factor involved in the developmental activation of the gene encoding the tissue specific transcription factor HNF1 alpha (LFB1).

The tissue specific transcription factor HNF1 alpha (LFB1) expressed in liver, kidney, stomach and gut gets transcriptionally activated in Xenopus shortly after zygotic transcription starts. By microinjection into fertilized Xenopus eggs, a HNF1 alpha promoter fragment is activated in the middle part of developing larvae, reflecting the activation pattern of the endogenous HNF1 alpha gene. Mutational analysis of the HNF1 alpha promoter shows that HNF1 and HNF4 binding sites are essential for proper embryonic regulation. Since by injecting HNF4 mRNA into fertilized eggs the endogenous HNF1 alpha gene is activated ectopically and HNF4 is present as a maternal protein within an animal to vegetal gradient in the embryo, we assume that HNF4 initiates a transcriptional hierarchy involved in determination of different cell fates.

Amino Acid Sequence↗

Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1.

The Arabidopsis GCN5, ADA2a and ADA2b proteins are homologs of components of several yeast and animal transcriptional coactivator complexes. Previous work has implicated these plant coactivator proteins in the stimulation of cold-regulated gene expression by the transcriptional activator protein CBF1. Surprisingly, protein interaction studies demonstrate that the DNA-binding domain of CBF1 (and of a related protein, TINY), rather than its transcriptional activation domain, can bind directly to the Arabidopsis ADA2 proteins. The ADA2a and ADA2b proteins can also bind directly to GCN5 through their N-terminal regions (comparable to a region previously defined in yeast Ada2) and through previously unmapped regions in the middle of the ADA2 proteins, which bind to the HAT domain of GCN5. The ADA2 proteins enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs. We speculate that this modification may represent a novel autoregulatory mechanism for the plant SAGA-like transcriptional coactivator complexes.

Acetylation↗

Age-associated shifts in cardiac gene transcription and transcriptional responses to ischemic stress.

Aged hearts exhibit reduced tolerance to ischemia-reperfusion, together with altered structure and post-ischemic remodelling. The molecular bases of such changes are unclear. Using cDNA microarrays and quantitative RT-PCR we characterized shifts in gene expression patterns with aging in normoxic and post-ischemic (20 min global ischemia, 60 min reperfusion) murine hearts (young: 2-4 months; aged: 16-18 months). We identified an age-associated up-regulation of transcripts involved in cell death, oxygen transport and metabolism in normoxic hearts. Down-regulated transcripts were involved in transporter activity, protein binding and hydrolase activity, changes in MAPK, WNT and TGF-beta signalling with aging were also observed. Ischemic stress generated a much greater degree of contractile impairment and cellular damage in aged vs. young hearts. This was associated with a substantially modified transcriptional response, with selective changes in Ca2+, WNT, NOTCH and G-protein coupled receptor signalling paths in aged vs. young hearts. Despite some common responses to ischemia in young and aged hearts (induction of heat shock protein transcripts), aging selectively modified ischemic responses of immediate early genes, and genes involved in modulating apoptosis and remodelling/angiogenesis. In summary, aging is associated with shifts in cardiovascular gene expression consistent with the phenotypic features of older hearts. Reduced tolerance with age may be related to modification of signalling (particularly WNT and TGF-beta), and shifts in expression of immediate early genes, and genes important in control of cell death/survival, angiogenesis, and cardiac remodelling.

Aging↗

A light-sensitive mechanism differently regulates transcription and transcript stability of omega3 fatty-acid desaturases (FAD3, FAD7 and FAD8) in soybean photosynthetic cell suspensions.

The omega3 fatty-acid desaturases: FAD7 and FAD8 (plastid) and FAD3 (reticular) are responsible for trienoic fatty-acid (TA) production in plants. The expression of these enzymes seemed to be regulated differently in response to light. Darkness leads to a decrease in total TA level. Under such conditions, FAD3 and FAD8 transcript levels were undetectable but increased after re-illumination concomitant with TA levels, indicating a transcriptional control. On the contrary, FAD7 transcript levels were similar to illuminated control cells, suggesting the presence of a post-transcriptional control mechanism. Furthermore, FAD7 mRNA stability increased dramatically in darkness. Analysis of FAD7 protein accumulation using specific antibodies revealed that FAD7 was very stable whatever the light or darkness conditions. These results indicate that FAD7 enzyme availability is not limiting for 18:3 production in darkness. Our data point to an additional post-translational regulatory mechanism that controls the activity of FAD7 in response to light.

Amino Acid Sequence↗

Transcriptional and post-transcriptional regulation of the PKC delta gene by etoposide in L1210 murine leukemia cells: implication of PKC delta autoregulation.

Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.

Acetophenones↗

Transcription coactivator CBP has direct DNA binding activity and stimulates transcription factor DNA binding through small domains.

CBP and p300 are transcriptional coactivators that physically interact with diverse sequence-specific DNA-binding factors through conserved domains. To further investigate the functional roles of these protein-interaction domains in CBP/p300 regulation, we have identified multiple domains of CBP that interact with FKLF2 and the CH2 domain as a new p53 interacting domain of CBP. Functional studies demonstrate that several domains of CBP are capable of stimulating FKLF2 and p53 DNA binding. In addition, we found that CBP through distinct domain is able to bind DNA directly with no specificity. We identified a 51-residue domain in CBP that is capable of interacting with both transcription factors and DNA. We named this domain PDBD for protein and DNA binding domain. These results unveiled two novel activities of CBP. First, these highly conserved domains of CBP not only function to recruit CBP to the target promoter through interaction with DNA-bound transcription factors, but they also actively regulate the DNA binding activity of their interacting factors. Second, by directly interacting with DNA, CBP may orchestrate the formation of stable and promoter-committed transcriptional complexes through interactions with both proteins and promoter DNA.

Amino Acid Sequence↗

Read-through histone transcripts containing 3' adenylate tails are zygotically expressed in Xenopus embryos and undergo processing to mature transcripts when introduced into oocyte nuclei.

Messages encoding replication-dependent histone genes generally terminate with a stem-loop structure and lack polyadenylate tails. Adenylated histone transcripts were identified in Xenopus oocytes, though the role of the adenylate tracts is unknown. We report isolation of cDNAs from Xenopus embryos encoding histone mRNAs with 3' adenylate tracts. They also contain targets for stem-loop binding protein and U7 snRNA, which are required for histone RNA processing. One sequence is a read-through transcript containing a complete version of the downstream gene from the anti-parallel strand, similar to the RNAs from lampbrush loops of Notophthalmus oocytes. We injected read-through transcripts into Xenopus oocyte nuclei and they were processed to mature histone RNAs. Our results suggest that addition of 3' adenylate sequences might be a normal part of histone RNA synthesis. Also, these results shed light on the enigma of the developmental regulation of adenylated histone transcripts in Xenopus oocytes.

Amino Acid Sequence↗

Cyclic AMP as a transcriptional inhibitor of upper eukaryotic gene transcription.

Recently, glucagon and its second messenger, cyclic AMP, have been shown to stimulate the transcription rate of several upper eukaryotic genes (1-5). We show here that glucagon can also block gene transcription. Both glucagon and cyclic AMP were found to inhibit the transcription of the genes encoding three liver glycolytic enzymes, including L-type pyruvate kinase and aldolase B. Thus, cyclic AMP proves to be not only an activator but also an inhibitor of gene transcription in eukaryotes.

Animals↗

A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination.

Bacteriophages↗

Transcriptional and post-transcriptional control of cold-shock genes.

A mesophile like Escherichia coli responds to abrupt temperature downshifts (e.g. from 37 degrees C to 10 degrees C) with an adaptive response that allows cell survival and eventually resumption of growth under the new unfavorable environmental conditions. During this response, bulk transcription and translation slow or come to an almost complete stop, while a set of about 26 cold-shock genes is preferentially and transiently expressed. At least some of the proteins encoded by these genes are essential for survival in the cold, but none plays an exclusive role in cold adaptation, not even the "major cold-shock protein" CspA and none is induced de novo. The majority of these proteins binds nucleic acids and are involved in fundamental functions (DNA packaging, transcription, RNA degradation, translation, ribosome assembly, etc.). Although cold-induced activation of specific promoters has been implicated in upregulating some cold-shock genes, post-transcriptional mechanisms play a major role in cold adaptation; cold stress-induced changes of the RNA degradosome determine a drastic stabilization of the cold-shock transcripts and cold shock-induced modifications of the translational apparatus determine their preferential translation in the cold. This preferential translation at low temperature is due to cis elements present in the 5' untranslated region of at least some cold-shock mRNAs and to trans-acting factors whose levels are increased substantially by cold stress. Protein CspA and the three translation initiation factors (IF3 in particular), whose stoichiometry relative to the ribosomes is more than doubled during the acclimation period, are among the trans elements found to selectively stimulate cold-shock mRNA translation in the cold.

Bacterial Proteins↗

Involvement of transcription termination factor 2 in mitotic repression of transcription elongation.

All nuclear transcription is interrupted during mitosis. We examined the role of human TTF2, an RNA polymerase (Pol) I and II termination factor, in mitotic repression of transcription elongation. We find that TTF2 levels rise in the cytoplasm in S and G2 and at the onset of mitosis TTF2 translocates into the nucleus. Consistent with a role in termination of all transcription, TTF2 is the only ATP-dependent termination activity associated with Pol II transcription elongation complexes, is largely unaffected by template position, and is impervious to the phosphorylation state of the polymerase. Cells in which TTF2 levels are knocked down showed dramatic retention of Ser2 phosphorylated Pol II on mitotic chromosomes and an increase in chromosome segregation defects.

Active Transport, Cell Nucleus↗

Measurements of human papillomavirus transcripts by real time quantitative reverse transcription-polymerase chain reaction in samples collected for cervical cancer screening.

Specific assays capable of distinguishing normal and atypical cervical changes from pre-cancerous lesions are direly needed to improve screening for cervical cancer. Specific genes transcripts that are up-regulated in dysplastic and cancer cells can be exploited as new markers for cervical cancer screening provided that they can be detected in heterogeneous populations such as those collected for Papanicolaou tests. We hypothesized that expression of the HPV early region gene E7 might distinguish between normal samples (absent expression) and high-grade lesions (detectable E7 expression). Our goal was to detect and measure gene expression in cells scraped from the cervix using real time quantitative reverse transcription-polymerase chain reaction (TaqMan). We have optimized collection and extraction procedures to provide suitable RNA for TaqMan analysis in clinical samples collected for cervical cancer screening and have demonstrated efficient measurements of housekeeping genes in these samples. HPV 16 or 18 early gene E7 transcripts were detected in 47% of samples with a clinical diagnosis of high-grade SIL and in 0% of cytologically normal samples (P = 0.006). Our study demonstrates that the TaqMan assay can be reliably applied to samples collected for cervical cancer screening, and that presence of detectable HPV E7 transcripts can distinguish between normal and abnormal samples.

DNA, Viral↗

Tumor cell contact mediated transcriptional activation of the fibroblast matrix metalloproteinase-9 gene: involvement of multiple transcription factors including Ets and an alternating purine-pyrimidine repeat.

The 92-kDa type IV collagenase (MMP-9) is a metalloproteinase frequently localized in both tumor stroma and in tumor cells, particularly at the tumor invasion front. To explore the factors regulating transcriptional activation of MMP-9 in stromal cells, we used a model system in which fibroblast MMP-9 expression can be upregulated by cell-cell contact with metastatic transformed rat embryo cells. Using transient transfection of reporter gene constructs containing 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, the upstream NFkappaB, SP-1, and Ets sites and the downstream AP-1 site and retinoblastoma binding element were shown to be necessary for basal transcriptional activity of fibroblast MMP-9. In contrast only Ets or SP-1 appeared to be involved in contact-mediated induction of MMP-9. Mutation of the upstream AP-1 site increased both basal and contact-stimulated promoter activation. Deletion of the alternating purine-pyrimidine repeat in the downstream promoter decreased transcriptional activity. Together these findings suggest that Ets and SP-1 are the central transcriptional activators of MMP-9 gene expression in fibroblasts specifically responding to tumor cell contact, and that promoter conformation may regulate MMP-9 expression.

Animals↗

Zonal distribution of transcripts of four hepatic transcription factors in the mature rat liver.

Within the liver acinus the majority of genes are expressed in an ascending or descending gradient from the portal to the central vein. The regulatory factors involved in this spatial pattern of gene expression are still poorly understood. Many liver genes are regulated by liver-enriched transcription factors. Here we report on mRNA distribution of four transcription factors in cell lysates obtained from either the periportal or the perivenous region after zone-specific digitonin infusion and by in-situ hybridization. Northern blot analysis revealed that there were slightly more transcripts of C/EBP, HNF1 beta (n.s.) and HNF4 (p < 0.05), but fewer of HNF3 gamma (n.s.), in perivenous than in periportal lysates. A somewhat stronger staining in the perivenous region of HNF4 was also seen by in-situ hybridization. The moderate acinar zonation of the mRNAs of these transcription factors suggests that at best they could modulate but not govern the zonated expression of liver genes in the mature liver.

Animals↗

Ectopic expression of the ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription.

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

Animals↗

Transcription repression of a CCAAT-binding transcription factor CBF/HSP70 by p53.

NF-Y transcription factor binds to CCAAT boxes on promoters of cell cycle regulatory genes such as cdc2, cyclin B, cdc25C, and cyclin A. We previously reported that the DNA binding activity of NF-Y is regulated by p53-p21-cdk2 pathway. CBF/HSP70 was originally identified as a transcription factor binding to the CCAAT box on the hsp70 promoter and mediates transcription repression of hsp70 pro- moter by p53. Recently it was demonstrated that CBF/HSP70 interacts and cooperates with NF-Y. In this study, we found that p53 represses the trans-cription of CBF/HSP70. Since transactivation ability of NF-Y is regulated in a cell cycle-dependent manner, we examined the transcription of CBF/HSP70 during the cell cycle. After synchronization of a human bladder carcinoma cell lacking functional p53 at early S phase, we infect the cells with adenovirus encoding p53. Cells infected with control virus progressed to S and G2 after release from the arrest. In contrast, cells expressing p53 enter S and G2 phases, but arrest at G2/M. The expression of CBF/HSP70 was induced at S/G2 phase in cells infected with a control virus, but kept to be repressed in cells expressing p53. Thus, these results suggest that p53 suppresses the expression of cell cycle regulatory genes though inhibiting both CCAAT binding factors, CBF/HSP70 and NF-Y.

CCAAT-Binding Factor↗

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5' part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

Base Sequence↗