Genomic organization of nucleolin gene in carp fish: Evidence for several genes

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602006000200017&lng=es&nrm=isoThe protein nucleolin, functionally involved in the main steps of ribosome biogenesis, is codified by a single copy gene in mammals. Here we report that at least three different genes codify for this protein in carp fish (Cyprinus carpio). This is the first description of the genomic organization of nucleolin in a teleost. The carp nucleolin gene includes 8.8 kb and contains 16 exons. Promoter cis regulatory elements are similar to constitutive genes, i.e., a putative TATA box, three G/C boxes, and three pyrimidine-rich boxes. As in other species, carp nucleolin gene introns host three snoRNA codifying sequences: U23 from the H/ACA family and two C/D box snoRNAs, U20 and U82. Both U20 and U82 span a complementary sequence with carp 18S rRNA. Additionally, we identified two cDNAs coding for nucleolin, confirming the existence of several nucleolin genes in carp. Amino acidderived sequence from carp cDNAs differ from mammal protein because they span additional acidic domains at the amino end, whose functional significance remains unclear. We performed amino acid sequence comparison and phylogenetic analyses showing that the three isoforms of carp nucleolin, which we describe herein, cluster in two groups. cNUC1 probably diverges from cNUC2 and cNUC3 as result of ancestral fish-specific genome duplication, indeed C. carpio is a tetraploid fish

A nucleosome-free dG-dC-rich sequence element promotes constitutive transcription of the essential yeast RIO1 gene

RIO1 is an essential gene that encodes a protein serine kinase and is transcribed constitutively at a very low level. Transcriptional activation of RIO1 dispenses with a canonical TATA box as well as with classical transactivators or specific DNAbinding factors. Instead, a dGdCrich sequence element, that is located 40 to 48 bp upstream the single site of mRNA initiation, is essential and presumably constitutes the basal promoter. In addition, we demonstrate here that this promoter element comprises a nucleosomefree gap which is centered at the dGdC tract and flanked by two positioned nucleosomes. This element is both, necessary and sufficient, for basal transcription initiation at the RIO1 promoter and, thus, constitutes a novel type of core promoter element

The RNA Polymerase II Core Promoter in Drosophila.

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in Drosophila The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-l, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity, The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication

Transcriptional regulation of the hepatitis B virus large surface antigen gene

Hepatitis B virus (HBV) is a hepatotropic virus of highly restricted host range and tissue specificity. Although the mechanisms governing this tropism are not fully understood, it is likely that restrictions occur at multiple steps in the viral life cycle. The liver-specific regulation of HBV gene expression suggests that transcription may be an important factor in the hepatotropism of the virus. An analysis of tissue- or cell- line-specific regulation of the HBV promoters may elucidate the role of transcriptional regulation in the hepatotropism of the virus. The major aim of this project was to characterize the transcriptional regulation of the large surface antigen gene of hepatitis B virus. To achieve this, the regions of the HBV genome involved in the regulation of the expression of the large surface antigen gene were identified using a transient transfection system in mammalian cell lines. The transcriptional activities of the four HBV promoters were compared in the human differentiated hepatoma cell lines Hep3B, PLC/PRF/5, HepG2 and Huh7, a human dedifferentiated hepatoma cell line HepG2.1, and the nonhepatoma cell lines HeLa S3 and NIH 3T3. To determine the relative transcriptional activities of the four HBV promoters, reporter gene plasmids were generated such that the expression of the firefly luciferase gene was under the control of each of the HBV promoters in the context of the complete genome. The nucleocapsid promoter and large surface antigen promoter displayed higher relative activities in the differentiated hepatoma cell lines, indicating that these promoters are preferentially active in these cell lines. A series of large surface antigen promoter deletion plasmids were constructed to identify the important regulatory regions of the large surface antigen promoter. The deletion analysis demonstrated that the region responsible for the high relative activity in differentiated hepatoma cell lines is located between -90 and -76 relative to the transcription initiation site (-*■!) located at map position 2809. This sequence element contains the binding site (GTTAATCATTACT) for the liver-enriched transcription factor hepatocyte nuclear factor I, HNF1. A eukaryotic expression vector containing the HNFI cDNA under the control of the mouse metallothionein I promoter was cotransfected with the HBV promoter constructs in Huh7 and HepG2.1 cells, and the relative levels of activity were determined. The Huh7 cell line was used because it is one of the cell lines in which HBV replication and particle production can occur and may represent the tissue culture system closest to the natural environment for the HBV life cycle, the liver cell. The cloned transcription factor HNFI activated transcription from the large surface antigen promoter, but not from any of the other HBV promoters. Cotransfection experiments using the HNFI cDNA expression vector and large surface antigen promoter deletion constructs demonstrated that this transactivation was mediated through the HNFI binding site located between -90 and -76 in the large surface antigen promoter. A series of deletion mutants of the cDNA in the HNFI expression vector was generated to determine the transcriptional activation domain of the HNFI polypeptide. The major domain of the HNFI polypeptide involved in transcriptional activation of the large surface antigen promoter in the human hepatoma cell line HepG2.1 was mapped to a region rich in glutamine and proline residues (9 of 18 residues). To demonstrate directly that the HNFI polypeptide produced by the expression of the HNFI cDNA could bind the large surface antigen promoter HNFI recognition sequence, and to determine whether a protein present in the differentiated hepatoma cell line Huh7 bound the HNFI element, gel mobility shift analysis was performed. This analysis demonstrated that a protein present in nuclear extracts from Huh7 cells formed a specific complex with the HNFI binding site which had similar migration properties to the complex formed between exogenously expressed HNFI and the HNFI recognition sequence. DNase I footprinting analysis demonstrated the binding of a protein present in the differentiated hepatoma cell line Huh7 to the HNFI recognition sequence in the large surface antigen promoter. DNase I footprinting also showed that purified TATA binding protein binds the TATA box element located between -31 and -23 in the large surface antigen promoter. The analysis of synthetic promoter constructs suggested that the HNFI and TATA box elements were the only elements necessary for maximal activity from the large surface antigen promoter, and analysis of clustered point mutations in the large surface antigen minimal promoter region demonstrated that sequences between the HNFI and TATA box elements were not required for the HNF1- dependent activity of the large surface antigen promoter. These studies suggested that the liver-enriched transcription factor HNFI plays a critical role in the cell-line and tissue-specific regulation of the HBV large surface antigen promoter

Identifying DNA motifs based on match and mismatch alignment information

The conventional way of identifying DNA motifs, solely based on match alignment information, is susceptible to a high number of spurious sites. A novel scoring system has been introduced by taking both match and mismatch alignment information into account. The mismatch alignment information is useful to remove spurious sites encountered in DNA motif searching. As an example, a correct TATA box site in Homo sapiens H4/g gene has successfully been identified based on match and mismatch alignment information

Wide-Scale Analysis of Human Functional Transcription Factor Binding Reveals a Strong Bias towards the Transcription Start Site

We introduce a novel method to screen the promoters of a set of genes with shared biological function, against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. The gene sets were obtained from the functional Gene Ontology (GO) classification; for each set and motif we optimized the sequence similarity score threshold, independently for every location window (measured with respect to the TSS), taking into account the location dependent nucleotide heterogeneity along the promoters of the target genes. We performed a high throughput analysis, searching the promoters (from 200bp downstream to 1000bp upstream the TSS), of more than 8000 human and 23,000 mouse genes, for 134 functional Gene Ontology classes and for 412 known DNA motifs. When combined with binding site and location conservation between human and mouse, the method identifies with high probability functional binding sites that regulate groups of biologically related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were put to several experimental tests. By allowing a "flexible" threshold and combining our functional class and location specific search method with conservation between human and mouse, we are able to identify reliably functional TF binding sites. This is an essential step towards constructing regulatory networks and elucidating the design principles that govern transcriptional regulation of expression. The promoter region proximal to the TSS appears to be of central importance for regulation of transcription in human and mouse, just as it is in bacteria and yeast.Comment: 31 pages, including Supplementary Information and figure