Downstream Core Promoter Element Research Articles

Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter: IDENTIFICATION OF CORE PROMOTER ELEMENTS AND FUNCTIONAL NUCLEAR FACTOR I-BINDING SITES

Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5'-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from -78 to -51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5' Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region -709/-598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment -612/-580, which contains a nuclear factor I (NFI)-binding site at the region -594/-580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at -147/-133, were identified in the LO promoter region -804/-1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation.

Perspectives on the RNA polymerase II core promoter

The RNA polymerase II core promoter is a critical yet often overlooked component in the transcription process. The core promoter is defined as the stretch of DNA, which encompasses the RNA start site and is typically approx. 40-50 nt in length, that directs the initiation of gene transcription. In the past, it has been generally presumed that core promoters are general in function and that transcription initiation occurs via a common shared mechanism. Recent studies have revealed, however, that there is considerable diversity in core promoter structure and function. There are a number of DNA elements that contribute to core promoter activity, and the specific properties of a given core promoter are dictated by the presence or absence of these core promoter motifs. The known core promoter elements include the TATA box, Inr (initiator), BRE(u) {BRE [TFIIB (transcription factor for RNA polymerase IIB) recognition element] upstream of the TATA box} and BRE(d) (BRE downstream of the TATA box), MTE (motif ten element), DCE (downstream core element) and DPE (downstream core promoter element). In this paper, we will provide some perspectives on current and future issues that pertain to the RNA polymerase II core promoter.

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, we find that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. Our analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on our results, we propose that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID.

Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs.

BackgroundThe canonical core promoter elements consist of the TATA box, initiator (Inr), downstream core promoter element (DPE), TFIIB recognition element (BRE) and the newly-discovered motif 10 element (MTE). The motifs for these core promoter elements are highly degenerate, which tends to lead to a high false discovery rate when attempting to detect them in promoter sequences.ResultsIn this study, we have performed the first analysis of these core promoter elements in orthologous mouse and human promoters with experimentally-supported transcription start sites. We have identified these various elements using a combination of positional weight matrices (PWMs) and the degree of conservation of orthologous mouse and human sequences – a procedure that significantly reduces the false positive rate of motif discovery. Our analysis of 9,010 orthologous mouse-human promoter pairs revealed two combinations of three-way synergistic effects, TATA-Inr-MTE and BRE-Inr-MTE. The former has previously been putatively identified in human, but the latter represents a novel synergistic relationship.ConclusionOur results demonstrate that DNA sequence conservation can greatly improve the identification of functional core promoter elements in the human genome. The data also underscores the importance of synergistic occurrence of two or more core promoter elements. Furthermore, the sequence data and results presented here can help build better computational models for predicting the transcription start sites in the promoter regions, which remains one of the most challenging problems.

The MTE, a new core promoter element for transcription by RNA polymerase II.

The core promoter is the ultimate target of the vast network of regulatory factors that contribute to the initiation of transcription by RNA polymerase II. Here we describe the MTE (motif ten element), a new core promoter element that appears to be conserved from Drosophila to humans. The MTE promotes transcription by RNA polymerase II when it is located precisely at positions +18 to +27 relative to A(+1) in the initiator (Inr) element. MTE sequences from +18 to +22 relative to A(+1) are important for basal transcription, and a region from +18 to +27 is sufficient to confer MTE activity to heterologous core promoters. The MTE requires the Inr, but functions independently of the TATA-box and DPE. Notably, the loss of transcriptional activity upon mutation of a TATA-box or DPE can be compensated by the addition of an MTE. In addition, the MTE exhibits strong synergism with the TATA-box as well as the DPE. These findings indicate that the MTE is a novel downstream core promoter element that is important for transcription by RNA polymerase II.

The DPE, a core promoter element for transcription by RNA polymerase II.

The core promoter is an important yet often overlooked component in the regulation of transcription by RNA polymerase II. In fact, the core promoter is the ultimate target of action of all of the factors and coregulators that control the transcriptional activity of every gene. In this review, I describe our current knowledge of a downstream core promoter element termed the DPE, which is a TFIID recognition site that is conserved from Drosophila to humans. The DPE is located from +28 to +32 relative to the +1 transcription start site, and is mainly present in core promoters that lack a TATA box motif. Moreover, in Drosophila, the DPE appears to be about as common as the TATA box. There are distinct mechanisms of basal transcription from DPE- versus TATA-dependent core promoters. For instance, NC2/Dr1-Drap1 is a repressor of TATA-dependent transcription and an activator of DPE-dependent transcription. In addition, DPE-specific and TATA-specific transcriptional enhancers have been identified. These findings further indicate that the core promoter is an active participant in the regulation of eukaryotic gene expression.

The DPE, a conserved downstream core promoter element that is functionally analogous to the TATA box.

Transcription by RNA polymerase II is a fundamentaland important biological process. There are tens of thousands of diverse genes that are transcribed in a highly regulated manner by the RNA polymerase II transcriptionalmachinery, and thus the mechanisms by which transcription is controlled are necessarily complex (for reviews,see Conaway and Conaway 1993; Smale 1994; Koleskeand Young 1995; Zawel and Reinberg 1995; Björklundand Kim 1996; Kaiser and Meisterernst 1996; Orphanides et al. 1996; Reines et al. 1996; Roeder 1996;Svejstrup et al. 1996; Verrijzer and Tjian 1996)...

The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.

We analyzed the function of the downstream promoter element (DPE), a distinct 7-nucleotide core promoter element that is approximately 30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters in Drosophila. There is a strict requirement for spacing between the Inr and DPE motifs, as an increase or decrease of 3 nucleotides in the distance between the Inr and DPE causes a seven- to eightfold reduction in transcription as well as a significant reduction in the binding of purified TFIID. These results suggest a specific and somewhat rigid interaction of TFIID with the Inr and DPE sequences. Photo-cross-linking analysis of purified TFIID with a TATA-less DPE-containing promoter revealed specific cross-linking of dTAFII60 and dTAFII40 to the DPE, with a higher efficiency of cross-linking to dTAFII60 than to dTAFII40. These data, combined with the previously well-characterized interactions between the two TAFs and their homology to histones H4 and H3, suggest that a dTAFII60-dTAFII40 heterotetramer binds to the DPE. Human and Drosophila transcription factors exhibit essentially the same requirements for DPE sequence and for Inr-DPE spacing. In addition, the TATA-less promoter of the human interferon regulatory factor-1 (IRF-1) gene contains a DPE that is important for transcriptional activity both in vitro and in cultured cells. Hence, these studies provide evidence for a direct role of TAFs in basal transcription of TATA-less DPE-containing genes and collectively indicate that the DPE is, in many respects, a downstream counterpart to the TATA box that is present in Drosophila to humans.