P1 Transcription Start Site Research Articles

Damage-inducible intragenic demethylation of the human TP53 tumor suppressor gene is associated with transcription from an alternative intronic promoter.

Wild‐type TP53 exons 5–8 contain CpG dinucleotides that are prone to methylation‐dependent mutation during carcinogenesis, but the regulatory effects of methylation affecting these CpG sites are unclear. To clarify this, we first assessed site‐specific TP53 CpG methylation in normal and transformed cells. Both DNA damage and cell ageing were associated with site‐specific CpG demethylation in exon 5 accompanied by induction of a truncated TP53 isoform regulated by an adjacent intronic promoter (P2). We then synthesized novel synonymous TP53 alleles with divergent CpG content but stable encodement of the wild‐type polypeptide. Expression of CpG‐enriched TP53 constructs selectively reduced production of the full‐length transcript (P1), consistent with a causal relationship between intragenic demethylation and transcription. 450K methylation comparison of normal (TP53‐wildtype) and cancerous (TP53‐mutant) human cells and tissues revealed focal cancer‐associated declines in CpG methylation near the P1 transcription start site, accompanied by rises near the alternate exon 5 start site. These data confirm that site‐specific changes of intragenic TP53 CpG methylation are extrinsically inducible, and suggest that human cancer progression is mediated in part by dysregulation of damage‐inducible intragenic CpG demethylation that alters TP53 P1/P2 isoform expression. © 2015 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

NF-YC Complexity Is Generated by Dual Promoters and Alternative Splicing

The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB, and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunits, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell-dependent; only one major NF-YC isoform is present in a given cell type. The 37- and 50-kDa isoforms are mutually exclusive, and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-PCRs indicate that P1 has a robust housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage in a p53-dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50-kDa transcript being excluded from P2. Specific functional inactivation of the 37-kDa isoform affects the basal levels of G(1)/S blocking and pro-apoptotic genes but not G(2)/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37-kDa isoform in suppression of the DNA damage-response under growing conditions.

Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control

Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.

Differential expression of Myc1 and Myc2 isoforms in cells transformed by eIF4E: evidence for internal ribosome repositioning in the human c-myc 5'UTR.

eIF4E is essential for translation initiation, but its overexpression causes malignant transformation. Recent work demonstrated that eIF4E/F participates in exposing and locating alternate translation start codons during scanning. Translation initiation of several important protooncogenes and growth-regulators, such as Myc and FGF-2, can start at CUG start codon(s) upstream of the normal open reading frame (ORF). The resulting amino-terminal extension alters the properties of these proteins and their intracellular distribution. In cells overexpressing eIF4E, c-myc is overexpressed and particularly the larger, CUG-initiated form (Myc1). Recent reports suggest that synthesis of Myc2, the normally expressed AUG-initiated form, is mediated by an IRES. To determine what role eIF4E might play in c-myc expression, the c-myc 5' untranslated region (UTR) was fused in-frame to CAT reporters, and several more derivative constructs were made. In vitro translation experiments (with and without eIF4E/F); expression in CHO cells transformed with eIF4E; and deletion/mutation analysis demonstrated that Myc1 is translated by a scanning mechanism, while Myc2 is translated by Internal Ribosome Repositioning. Moreover, the existence of a true IRES in the 5'UTR was contradicted by its failure to direct translation of a circular transcript, in contrast to hsp70. The c-myc 5'UTR also failed to engage in translation in the absence of functional eIF4F, after cleavage of the eIF4G component with CVB4 protease-2A. The Internal Repositioning Element (IRPE) in c-myc 5'UTR was delimited to nucleotides (nt) 394-440 from the P1 transcription start site.

Inhibition of nuclear protein binding to two sites in the murine c-myc promoter by intermolecular triplex formation.

The c-myc gene is overexpressed in a variety of tumor types and appears to play an important role in the abnormal growth of a number of cell types. In an effort to determine the ability of sequence- and species-specific triplex-forming oligonucleotides to inhibit expression of a targeted gene in animals, we have identified two novel triplex-forming sites in the murine c-myc promoter. One is homologous to the triplex-forming human PuF binding element located upstream of the P1 transcription start site. The other triplex-forming site is found in a region between P1 and P2 that encompasses the ME1a1 binding site and part of the E2F binding site and is highly homologous to the human sequence. Synthetic oligodeoxyribonucleotides designed to target these essential regulatory elements form sequence-specific triple helices as demonstrated by gel mobility shift analysis and DNase I footprinting. Polypurine: polypyrimidine regions in the P1 and P2 promoters form specific protein-DNA complexes upon incubation with a murine YC8 nuclear extract. Preincubation of each of the promoter fragments with its respective triplex-forming oligonucleotide results in the inhibition of nuclear protein binding. Non-triplex-forming oligonucleotides do not significantly affect protein binding. The data presented are a preliminary step toward generating an animal model for the phenotypic effects of triplex formation within the c-myc promoter.

Correlation between suppression of c-myc and antiproliferative effect of transforming growth factor-beta 1 in thyroid carcinoma cell growth.

The growth regulatory activity of transforming growth factor-beta 1 (TGF beta 1) was studied in a clonal strain of thyroid papillary carcinoma cell (NPA). Despite the presence of TGF beta 1 and its receptor messenger RNA in thyroid carcinoma, the molecular mechanism of TGF beta 1 action on cell growth of thyroid carcinoma has not yet been elucidated. Exogenously added TGF beta 1 inhibited DNA synthesis and cell growth in a dose- and time-dependent manner at concentrations of 0.1-10 ng/ml. TGF beta 1 inhibited not only basal but also fetal bovine serum-stimulated cell proliferation. Steady state levels of c-myc messenger RNA transcripts were inhibited by TGF beta 1 after 0.5-h treatment. Antisense, but not sense, c-myc oligodeoxynucleotides also caused suppression of NPA cell growth in a dose-responsive manner. Transfection studies of the 5'-up-stream flanking region (UFR) of c-myc/chloramphenicol acetyltransferase chimera genes suggest the presence of a TGF beta 1-responsive DNA element in the 2.3-kilobase c-myc 5'-UFR. Deletion mutant studies indicate the element lies between -106 to 70 relative to the P1 transcription start site. Studies with the gel mobility shift assay using 23-basepair double strand DNA showed the presence of at least two nuclear factors in NPA cell. TGF beta 1 treatment did not cause any alteration in TGF beta 1-induced mobility; however, the reduction of a positive band was selectively observed during 30 min to 2 h after treatment with TGF beta 1. In contrast, the position and intensity of another band were not altered by TGF beta 1 treatment. These results demonstrate that the inhibition of a nuclear factor binding to the c-myc 5'-UFR and subsequent suppression of c-myc gene expression are directly involved in the antiproliferative action of TGF beta 1 in NPA cell growth.

Evidence for a regulatory function of the histone-like Escherichia coli protein H-NS in ribosomal RNA synthesis.

We have isolated a small Escherichia coli protein which stably interacts with ribosomal RNA P1 promoter DNA. We present evidence showing that the protein is identical to the histone-like E. coli protein, H-NS (H1). Binding of H-NS to the P1 promoter region is dependent on the DNA curvature. Mapping the H-NS-DNA contact sites by nuclease protection and high-resolution footprinting techniques reveal three H-NS-binding domains, and contacts of the protein in the major groove of the bent DNA. The binding region extends from position -18 to -89, relative to the P1 transcription start site, and shows an overlap with the known binding sites for Fis, another E. coli protein, which acts as transcriptional activator of P1. The binding of H-NS does not displace Fis; instead, heterologous complexes are formed. Apparently, H-NS and Fis bind to separated curved DNA segments, with the planes of the curves pointing into different directions. In vitro transcriptional analyses demonstrate that H-NS represses rRNA P1 promoter-directed transcription. Repression is most pronounced in the presence of Fis. Thus, H-NS seems specifically to antagonize Fis-dependent activation. No comparable inactivation is observed for the second rRNA promoter P2.

Yin-yang 1 activates the c-myc promoter.

Previous studies on the murine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in murine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.

Yin-Yang 1 Activates the c-myc Promoter

Previous studies on the murine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in murine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.

Identification of a functional initiator sequence in the human MDR1 promoter

The sequence requirements for proper transcriptional initiation of the downstream human multidrug resistance MDR1 (P1) promoter were determined using a transient expression system in HeLa cells. The MDR1 promoter has no TATA box and the transcription start site has a strong homology with the initiator ( Inr) sequence identified in the murine terminal deoxynu-cleotidyltransferase ( TdT) gene. A deletion analysis showed that sequences from −6 to +11 relative to the P1 transcription start site were sufficient for proper transcriptional initiation, whereas deletion of sequences downstream of +11 resulted in a strong reduction of properly initiated transcripts. In this transient assay system, both the MDR1 and TdT initiator require in Hela cells the presence of an upstream activating sequence such as the SV40 enhancer. This is in contrast to the transcription in in vitro systems, in which the initiator sequence is able to direct transcription in the absense of an enhancer. Analysis of mutations in the initiator sequence from −8 to +10 showed that the A and T nucleotides at position +1 and +3, respectively, were essential, whereas other substitutions in this region had little effect on promoter activity.

The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

A major site of DNA bending is located 1.6 kb upstream of the P1 transcription start site of the human c-myc gene, near the center of a reported zone of initiation of DNA replication. A repeated, purine-rich element, termed PUR, at the bend site is specifically bound by a protein in HeLa cell nuclear extracts. This protein has specific affinity for the purine-rich single strand of the element. Methylation interference maps a pattern of specific contact points with guanosine bases in a 24-mer oligonucleotide containing the element. UV cross-linking reveals that contact is made by a polypeptide of approximately 28 kDa. The PUR element is present at origins of replication and in gene flanking regions in a variety of eukaryotes from yeasts through humans. The consensus sequence GGNNGAGGGAGARRRR has been derived. This element is present near centers of regions of two mammalian loci (human c-myc and hamster dhfr) recently reported as initiation zones for DNA replication. A 24-mer oligonucleotide representing the hamster dhfr version of the PUR element effectively competes with the human c-myc version for binding to Pur.

The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

A major site of DNA bending is located 1.6 kb upstream of the P1 transcription start site of the human c-myc gene, near the center of a reported zone of initiation of DNA replication. A repeated, purine-rich element, termed PUR, at the bend site is specifically bound by a protein in HeLa cell nuclear extracts. This protein has specific affinity for the purine-rich single strand of the element. Methylation interference maps a pattern of specific contact points with guanosine bases in a 24-mer oligonucleotide containing the element. UV cross-linking reveals that contact is made by a polypeptide of approximately 28 kDa. The PUR element is present at origins of replication and in gene flanking regions in a variety of eukaryotes from yeasts through humans. The consensus sequence GGNNGAGGGAGARRRR has been derived. This element is present near centers of regions of two mammalian loci (human c-myc and hamster dhfr) recently reported as initiation zones for DNA replication. A 24-mer oligonucleotide representing the hamster dhfr version of the PUR element effectively competes with the human c-myc version for binding to Pur.

Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product.

Previous studies have shown that transforming growth factor beta 1 (TGF-beta 1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-beta 1. The same c-myc promoter region was required for regulation by both TGF-beta 1 and pRB. These sequences, termed the TGF-beta control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-beta 1 treatment of TGF-beta-sensitive but not TGF-beta-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-beta 1 pathway for the inhibition of c-myc transcription and growth inhibition.

Transforming growth factor beta 1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation.

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types, including most epithelial cells. However, the mechanism of growth inhibition is unknown. In skin keratinocytes, TGF-beta 1 has been shown to inhibit growth and to rapidly reduce c-myc expression. It has been demonstrated that protein synthesis is required for TGF-beta 1 regulation of c-myc in keratinocytes. Here we present evidence that treatment of mouse BALB/MK keratinocyte cells with either antisense c-myc oligonucleotides or TGF-beta 1 inhibited cell entry into S phase. These results suggest that TGF-beta inhibition of c-myc expression may be essential for growth inhibition by TGF-beta 1. The block in c-myc expression by TGF-beta 1 occurred at the level of transcriptional initiation. Studies with a series of 5' deletion c-myc/chloramphenicol acetyltransferase constructs indicated that a cis regulatory element(s), which resides between positions -100 and +71 relative to P1 transcription start site, is responsible for the TGF-beta 1 responsiveness. Based on these data, it is proposed that the mechanism of TGF-beta 1 growth inhibition involves synthesis or modification of a protein that may interact with a specific element(s) in the 5' regulatory region of the c-myc gene, resulting in inhibition of transcriptional initiation.

A transcriptional repressor of c-myc.

In murine plasmacytomas there is deregulated transcription of a translocated c-myc allele and undetectable transcription of the normal, unrearranged c-myc allele. Deregulated c-myc transcription probably contributes to the transformed phenotype of the tumour cells, whereas repression of the normal allele probably reflects the normal turn-off of c-myc in non-dividing plasma cells. We previously identified a plasmacytoma-specific protein which binds to the c-myc promoter region 290 base pairs 5' of the P1 transcription start site. This plasmacytoma repressor factor (myc-PRF; formerly myc-PCF) is not found in cell lines representing earlier B-cell stages during which c-myc is transcribed, so it could be a negative regulator of c-myc transcription in terminally differentiated B cells. Here we report that site-directed deletion of the binding site for this protein leads to a 30-fold increase in transcription of a stably transfected c-myc fusion construct in plasmacytoma cells but has no effect in L cells or 18-81 pre-B cells, which lack the protein. Myc-PRF interacts with another widely distributed protein, myc-CF1 (common factor 1), which binds nearby, and this association may be important in myc-PRF repression.