Association of TATA box-binding protein-associated factor RNA polymerase I subunit C (TAF1C) with T2DM.

Proteins differential expression in type 2 diabetes mellitus (T2DM) can be due to etiological factors or pathological changes, such proteins can be utilized as biomarkers. Identification of a marker protein out of thousands became a feasible task during the proteomics era by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, blood samples were obtained from 80 Bahraini subjects with and without T2DM, a subset was used for proteomic analysis by LC-MS/MS, while all samples were used for ELISA analysis of 3 proteins, TATA-box binding protein-associated factor RNA polymerase-1-C (TAF1C), ceruloplasmin (CERP) and fibronectin (FN). The former 2 proteins were selected from the T2DM-protein-panel identified by LC-MS/MS, and the latter was analyzed for validation of the setting. The main findings of the proteomic analysis are i. Identifications of 62 differentially expressed proteins in T2DM, ii. Upregulation of 71% of the identified proteins. While the ELISA analysis showed that; both TAF1C and FN were significantly increased in T2DM (P0.015 and P0.001, respectively), while CERP was not (P0.088). Logistic regression analysis: i. confirmed the above associations after correction for covariates, ii. Revealed an interaction between age and gender that affect the association of the proteins with T2DM. In conclusion, knowing that TAF1C is a prerequisite in ribosomal biogenesis, our ELISA results are suggestive of increased protein synthesis in T2DM, explaining the observed upregulation of the proteins identified by LC-MSMS. The association between T2DM and TAF1C is a novel finding that might open a new avenue in DM research.

Identification of evolutionarily conserved downstream core promoter elements required for the transcriptional regulation of Fushi tarazu target genes.

The regulation of transcription initiation is critical for developmental and cellular processes. RNA polymerase II (Pol II) is recruited by the basal transcription machinery to the core promoter where Pol II initiates transcription. The core promoter encompasses the region from -40 to +40 bp relative to the +1 transcription start site (TSS). Core promoters may contain one or more core promoter motifs that confer specific properties to the core promoter, such as the TATA box, initiator (Inr) and motifs that are located downstream of the TSS, namely, motif 10 element (MTE), the downstream core promoter element (DPE) and the Bridge, a bipartite core promoter element. We had previously shown that Caudal, an enhancer-binding homeodomain transcription factor and a key regulator of the Hox gene network, is a DPE-specific activator. Interestingly, pair-rule proteins have been implicated in enhancer-promoter communication at the engrailed locus. Fushi tarazu (Ftz) is an enhancer-binding homeodomain transcription factor encoded by the ftz pair-rule gene. Ftz works in concert with its co-factor, Ftz-F1, to activate transcription. Here, we examined whether Ftz and Ftz-F1 activate transcription with a preference for a specific core promoter motif. Our analysis revealed that similarly to Caudal, Ftz and Ftz-F1 activate the promoter containing a TATA box mutation to significantly higher levels than the promoter containing a DPE mutation, thus demonstrating a preference for the DPE motif. We further discovered that Ftz target genes are enriched for a combination of functional downstream core promoter elements that are conserved among Drosophila species. Thus, the unique combination (Inr, Bridge and DPE) of functional downstream core promoter elements within Ftz target genes highlights the complexity of transcriptional regulation via the core promoter in the transcription of different developmental gene regulatory networks.

Nuclear respiratory factor 1 mediates the transcription initiation of insulin-degrading enzyme in a TATA box-binding protein-independent manner.

CpG island promoters often lack canonical core promoter elements such as the TATA box, and have dispersed transcription initiation sites. Despite the prevalence of CpG islands associated with mammalian genes, the mechanism of transcription initiation from CpG island promoters remains to be clarified. Here we investigate the mechanism of transcription initiation of the CpG island-associated gene, insulin-degrading enzyme (IDE). IDE is ubiquitously expressed, and has dispersed transcription initiation sites. The IDE core promoter locates within a 32-bp region, which contains three CGGCG repeats and a nuclear respiratory factor 1 (NRF-1) binding motif. Sequential mutation analysis indicates that the NRF-1 binding motif is critical for IDE transcription initiation. The NRF-1 binding motif is functional, because NRF-1 binds to this motif in vivo and this motif is required for the regulation of IDE promoter activity by NRF-1. Furthermore, the NRF-1 binding site in the IDE promoter is conserved among different species, and dominant negative NRF-1 represses endogenous IDE expression. Finally, TATA-box binding protein (TBP) is not associated with the IDE promoter, and inactivation of TBP does not abolish IDE transcription, suggesting that TBP is not essential for IDE transcription initiation. Our studies indicate that NRF-1 mediates IDE transcription initiation in a TBP-independent manner, and provide insights into the potential mechanism of transcription initiation for other CpG island-associated genes.

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box.

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P(2) promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment.

TBP2 is a substitute for TBP in Xenopus oocyte transcription.

BACKGROUND: TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos. RESULTS: We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription in vivo. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos. CONCLUSION: The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation.

An essential set of basic DNA response elements is required for receptor-dependent transcription of the lecithin:retinol acyltransferase (Lrat) gene.

Lecithin:retinol acyltransferase (LRAT) is essential for vitamin A storage. Nuclear run-on assays demonstrated transcriptional regulation of the Lrat gene in vivo by all-trans-retinoic acid (RA) and other retinoids. Analysis of a 2.5 kb segment of rat genomic DNA revealed that the region approximately 300 bp upstream from the transcription start site (TSS) is necessary for high luciferase (Luc) reporter activity in HEK293T and HepG2 cells. Although this region lacks retinoid receptor binding elements, it responded to the nuclear receptors RARalpha, RARbeta or RARgamma, with RXRalpha, with and without ligand. Removal of -111 bp from the TSS, which is well conserved in human, rat and mouse genomes, completely eliminated activity. This region contains several basic elements (TATA box, SP3 site, AP-1 site, CAAT box), all of which were essential. Nuclear extracts from RA-treated cells exhibited enhanced binding. Therefore, this proximal region together with basal transcription factors may be sufficient to drive Lrat expression.

Interactions between upstream and core promoter sequences determine gene expression and nucleosome positioning in tobacco PR-1a promoter.

The expression of PR-1a gene in tobacco is accompanied by changes in the chromatin architecture over its promoter region. The transcription initiates when the gene is induced in defense response, a condition that can be simulated experimentally by external application of salicylic acid. Mutagenesis of the core promoter sequence established that the TATA-box was critical to the expression of PR-1a gene. In order to study functional specificity between the core promoter and upstream activator region, the native core promoter was exchanged with that of a heterologous salicylic acid inducible promoter, Pcec. The core promoter and the activator region of PR-1a together determine its tightly regulated expression, slow kinetics of induction by SA and several fold induction of expression. In uninduced state, a single nucleosome was present over the core promoter of PR-1a. It masked both the TATA-box and the transcription initiation region. The transcriptional activation of the promoter by SA was accompanied by shift in the position of this nucleosome. The chimeric promoters failed to show inducibility or gave very low level of induction. They showed failure in shifting the nucleosome from the core promoter region. The promoter Pcec expressed constitutively at a high uninduced level in spite of a nucleosome over the TATA-box region. However, in this case, the nucleosome did not mask the transcript initiation region. The TATA-box nucleosome was shifted as the expression increased further, following induction by SA. A fully induced Pcec had the TATA-box fully exposed, though a weak nucleosome appeared on the +1 region. The results support a close relationship among promoter sequence architecture, nucleosome positioning and PR-1a expression.

Methylation of the prominin 1 TATA-less main promoters and tissue specificity of their transcript content.

Prominin 1 (PROM1, CD133) is a unique transmembrane glycoprotein encoded by the PROM1 gene. It is a cell surface marker of various stem cells including hematopoietic, prostatic epithelial, pancreatic, leukemic, liver cancer, and colorectal cancer stem cells. Here, we studied tissue specificity of PROM1 transcription isoforms and the methylation level of its two main promoters (P1 and P2) in different human cell lines. Only transcripts lacking the 4th exon (the CD133.s1 form) were expressed in cell lines studied. Moreover, these transcripts, if sufficiently abundant, were initiated simultaneously and independently from both promoters P1 and P2. In cell lines with low levels of the total PROM1 transcript, the transcription was likely initiated from other promoters. Promoter P1 was hypermethylated in all cell lines under study, and therefore, methylation can hardly play an important role in its regulation. In contrast, the methylation of promoter P2 was tissue specific, and hypomethylation of this promoter is probably necessary but not sufficient for efficient transcription of the PROM1 gene. Therefore, we report an unusual instance of different mechanisms of transcription activity regulation for two closely located promoters of the same gene.

Promoter influences transcription elongation: TATA-box element mediates the assembly of processive transcription complexes responsive to cyclin-dependent kinase 9.

Pausing of RNA polymerase II (RNAPII) during transcript elongation is an important mechanism for regulating gene expression at many genes. In this study we investigated the mechanism of regulated elongation of c-myc and human immunodeficiency virus-1 (HIV-1) using an in vitro elongation assay that reproduces the conditional block to elongation. We found that HIV-1 Tat can activate the RNAPII transcription complexes paused on c-myc by enhancing their elongation efficiency. We determined that cyclin-dependent kinase 9 (CDK9), the kinase subunit of positive transcription elongation factor b (P-TEFb) complex, regulates transcriptional elongation of c-myc and is present in transcription pre-initiation complexes formed on the c-myc promoter, which emphasizes a common mechanism of elongation control between HIV-1 and c-myc genes. We also investigated the roles of upstream elements of the HIV-1 and c-myc promoters in CDK9-activated transcriptional elongation. We found that the TATA-box element mediates the assembly of processive transcription complexes responsive to CDK9 and that specific combinations of upstream activation binding sites contribute to the recruitment of these complexes. We propose a common mechanism for elongation control at the c-myc and HIV-1 genes with an essential role for the TATA-box and specific modulatory contribution of upstream regulatory sequences, derived from the unique structure of the promoters, to form a composite surface for efficient recruitment of elongation-competent transcription complexes.

Noncanonical TATA sequence in the UL44 late promoter of human cytomegalovirus is required for the accumulation of late viral transcripts.

During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi, J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, "TATAA." In contrast, the UL44 late viral promoter has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.

[Nucleosome positioning within neomycinphosphotransferase gene (NPTII) on yeast plasmid in repressed and active state].

Yeast recombinant plasmid containing FRT-sequence flanked by hybrid GAL-CYC promoter and NPTII gene was developed. GAL-CYC promoter contains four UAS sequences and two closely associated TATA-boxes in CYC part. This construct provides galactose-inducible synthesis of neomycinphosphotransferase from NPTII gene, and, thus, resistance of transformed cells to G418 antibiotic. Nucleosome positioning within NPTII gene in repressed and active states was studied. Under repressive conditions (growth on glucose) stable positioning of three nucleosomes was detected. Two nucleosomes are localized in CYC-part. One of them encompasses both TATA-boxes. The third nucleosome overlaps FRT sequence and start of NPTII gene coding sequence. All three nucleosomes show multiple positioning. It suggests possibility of nucleosome sliding along DNA. After induction of NPTII expression by galactose sliding of two nucleosomes is detected. Sliding leads to exposure of TATA-box and long promoter segment. Sliding results in stable repositioning of nucleosomes at new sites. 5'-distal nucleosome moves closer to UAS-sequences. As a results UAS becomes spatially closer to TATA-box. This proximity facilitates assembly of preinitiation complex. Nucleosomes slides independently from each other. The second nucleosome moves towards FRT-sequence and repositions at its nucleosome positioning signal. Galactose-induced expression does not affect nucleosome positioning with coding region of NPTII gene. Unidirectional sliding and repositioning are detected without induction after deacetylase inhibition with trichostatine A. Basal expression of NPTII gene was shown without activation of GAL-CYC promoter and after spatial uncoupling of coding sequence and promoter by gene inversion. In these cases it seems that expression is driven by TATA-like element in FRT-sequence. This element is located in permanently exposed area (in vivo data).

Characterization and functional analysis of the promoter for the human equilibrative nucleoside transporter gene, hENT1.

Equilibrative nucleoside transporters (ENTs) are membrane proteins that transport nucleosides, nucleobases and analogs across membranes. ENT genes and the regulation of their expression are poorly understood. Therefore, we isolated and functionally characterized the promoter of the prototypic human ENT, hENT1. A single transcriptional initiation site 58 bp downstream of the TATA box and 272 bp upstream of the translation initiation site is present. Limited sequence similarity exists between the hENT1 and mouse ENT1 (mENT1) promoters suggesting conservation of ENT1 transcriptional regulators in mammals. Putative consensus sites for transcription factors exist within the hENT1 promoter. Reporter assays revealed similar but not identical transcriptional activity profiles in human cells.

The TATA-box sequence in the basal promoter contributes to determining light-dependent gene expression in plants.

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T(7) or A(8)-->C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants.

PU.1 and a TTTAAA element in the myeloid defensin-1 promoter create an operational TATA box that can impose cell specificity onto TFIID function.

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein ("D1BP"), acting through, respectively, proximal (-22/-19) and distal (-62/-59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)alpha/GABPbeta. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking "TTTAAA" element (-32/-27), a "weak" but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a "strong" TATA box ("TATAAA") eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.

Transcription regulation from a TATA and INR-less promoter: spatial segregation of promoter function.

The mode of regulation of class II genes that lack the known core promoter elements is presently unclear. Here, we studied one such example, the murine CD80 gene. An unusual mechanism was revealed wherein the pre-initiation complex (PIC) first assembled on an upstream, NF-kappaB enhancer element. Notably, this assembly occurred independent of contributions from the core promoter domain, and resulted in a PIC that was competent for transcription initiation. Positioning was subsequently achieved by exploiting the intrinsic architecture of the promoter, by virtue of which the tethered PIC was spatially juxtaposed with the transcription initiation site. Bridging interactions then ensued, through protein-protein contacts, which then enabled the elongation phase of CD80 transcription.

Role of the spacer between the -35 and -10 regions in sigmas promoter selectivity in Escherichia coli.

In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.

Transcriptional activation by bidirectional RNA polymerase II elongation over a silent promoter.

Transcriptional interference denotes negative cis effects between promoters. Here, we show that promoters can also interact positively. Bidirectional RNA polymerase II (Pol II) elongation over the silent human endogenous retrovirus (HERV)-K 18 promoter (representative of 2.5 x 10(3) similar promoters genomewide) activates transcription. In tandem constructs, an upstream promoter activates HERV-K 18 transcription. This is abolished by inversion of the upstream promoter, or by insertion of a poly(A) signal between the promoters; transcription is restored by poly(A) signal mutants. TATA-box mutants in the upstream promoter reduce HERV-K 18 transcription. Experiments with the same promoters in a convergent orientation produce similar effects. A small promoter deletion partially restores HERV-K 18 activity, consistent with activation resulting from repressor repulsion by the elongating Pol II. Transcriptional elongation over this class of intragenic promoters will generate co-regulated sense-antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome.

Identification of promoter elements in 5'-flanking region of murine cyclic nucleotide phosphodiesterase 3B gene.

We describe techniques for identifying functional promoter elements in the 5'-flanking region of the murine cyclic nucleotide phosphodiesterase 3B (mPDE3B) gene. The 5'-flanking region of the mPDE3B gene was cloned and sequenced, and putative transcription factor binding sites were identified with computational tools. A series of reporter plasmids containing the luciferase gene fused to different fragments of the 5'-flanking region of the mPDE3B gene was constructed and used to transfect 3T3-L1 fibroblasts or differentiating adipocytes. Reporter gene assays showed that there are two promoter regions in the 5'-flanking region in the mPDE3B gene: a distal region located approx 4 kb upstream of the translation initiation site that contains cAMP-response element (CRE) cis-acting elements, and a proximal region that is GC rich and lacks TATA sequences. The distal promoter region induced much higher luciferase activity than did the proximal one. Mutation of the CRE sequences or reversal of the orientation of the CRE-containing region abolished promoter activity of the distal region. Electrophoretic mobility shift assay analysis indicated that binding to CRE elements was greater in nuclear extracts from differentiating adipocytes than from fibroblasts. Mapping of transcription initiation sites suggested that the distal promoter region might function as an enhancer, whereas the proximal promoter drives transcription of the mPDE3B gene.