Functional TATA Box Research Articles

AI-driven designed protein epigenomics.

The biological revolutions of computationally designed proteins, induced pluripotent stem cells (iPSCs), and the CRISPR-Cas9 system finally enables modifications that can spur deep understanding of spatial requirement of epigenetic information. This commentary describes the utility of a computationally designed protein, EED Binder (EB), when fused to dCas9 (EBdCas9) for identifying critical sites of PRC2 dependent histone H3K27me3 marks in the chromatin. By using EBdCas9 and gRNA, PRC2 function can be inhibited at specific loci, resulting in precise reduction of EZH2 and H3K27me3 marks, and in some (but not all) locations, activation of the gene and functional outcomes (such as regulation of cell cycle or trophoblast transdifferentiation). Interestingly, a functional TATA box located more than 500bp upstream of a TBX18 TSS was found to be repressed by PRC2, supporting the theory that epigenetic regulators control the repression of transcriptional elements on the promoter region. The EBdCas9 technology may provide a useful tool for controlling gene regulation through epigenomic control.

DCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

SUMMARYBifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Computer Designed PRC2 Inhibitor, EBdCas9, Reveals Functional TATA Boxes in Distal Promoter Regions

The critical process in development, bifurcation of cellular fates, requires epigenetic H3K27me3 marks propagated by PRC2 complex. However, the precise chromatin loci of functional H3K27me3 marks are not yet known. Here we identify critical PRC2 functional sites at a single nucleosome resolution. We fused a computationally designed protein, EED binder (EB) that competes with EZH2 and thereby disrupts PRC2 function, to dCas9 (EBdCas9) to direct PRC2 inhibition at a precise locus using gRNA. We targeted EBdCas9 to 4 different genes (TBX18, p16, CDX2 and GATA3) and observed epigenetic remodeling at a single nucleosome resolution resulting in gene activation. Remarkably, while traditional TATA box is located 30bp upstream of TSS, we identified a functional TATA box, >500bp of TSS, normally repressed by PRC2 complex. Deletion of this TATA box eliminates EBdCas9 dependent TBP recruitment and transcriptional activation. Targeting EBdCas9 to CDX2 and GATA3 results in trophoblast trans-differentiation. EBdCas9 technology is broadly applicable for epigenetic regulation at a single locus to control gene expression.

Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

Distinct Role of Core Promoter Architecture in Regulation of Light-Mediated Responses in Plant Genes

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less (initiator-containing) promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr (Initiator) elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS (transcription start site), and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Regulation of the varicella-zoster virus ORF3 promoter by cellular and viral factors

The varicella zoster virus (VZV) immediate early 62 protein (IE62) activates most if not all identified promoters of VZV genes and also some minimum model promoters that contain only a TATA box element. Analysis of the DNA elements that function in IE62 activation of the VZV ORF3 promoter revealed that the 100 nucleotides before the translation start site of the ORF3 gene contains the promoter elements. This promoter lacks any functional TATA box element. Cellular transcription factors Sp1, Sp3 and YY1 bind to the promoter, and mutation of their binding sites inhibited ORF3 gene expression. VZV regulatory proteins, IE63 and ORF29, ORF61 and ORF10 proteins inhibited IE62-mediated activation of this promoter. Mutation of the Sp1/Sp3 binding site in the VZV genome did not alter VZV replication kinetics. This work suggests that Sp family proteins contribute to the activation of VZV promoters by IE62 in the absence of functional TATA box.

Gallstone disease in Swedish twins is associated with the Gilbert variant of UGT1A1

The Gilbert syndrome-associated functional TATA box variant UGT1A1*28 (A(TA)7TAA) was found to increase susceptibility to pigment gallstone formation in patients with haemolytic anaemia. Further studies in extensive cohorts demonstrated an increased risk of this variant for cholesterol gallstone disease (GD). We now investigated this polymorphism as a determinant of symptomatic GD in Swedish twins. The Swedish Twin Registry was merged with the Hospital Discharge and Causes of Death Registries and searched for GD-related diagnoses among monozygotic (MZ) twins living in the Stockholm area. In addition, we screened the TwinGene database for GD. In total, we found 44 MZ twin pairs with and eight MZ twins without GD to be evaluable. GD-free twins from TwinGene (109 concordantly MZ and 126 independent DZ) served as controls. UGT1A1*28 genotyping was performed using TaqMan assays. Overall, 58 and 8 of 106 twins with GD were hetero- and homozygous UGT1A1 risk allele carriers respectively. The case-control association tests showed a significantly (P < 0.05) increased risk of developing GD (OR = 1.62, 95% CI 1.00-2.63) in heterozygotes carriers and in addition, a trend (P = 0.075) for an increased risk among carriers (OR = 1.52, 95% CI 0.97-2.44) of the risk allele. These data from Swedish twins confirm the Gilbert variant as risk factor for GD. Our observation is in line with nucleation in bilirubin supersaturated bile representing an initial step in cholelithogenesis.

Gallstone disease is linked to the Gilbert-Meulengracht UGT1A1*28 variant: case-control analysis of Swedish twins

Background and Aims: The Gilbert-Meulengracht syndrome-associated functional TATA box variant UGT1A1*28 (A(TA)7TAA) increases susceptibility to pigment gallstone formation in the setting of haemolytic anemias [1]. Latest studies demonstrated that carriers of this variant bear an increased risk of cholesterol stones as well [2]. Here we investigate the UGT1A1 variant as a determinant of gallstone disease in a unique cohort of Swedish Twins.

A cluster of transcripts encoded by KSHV ORF30-33 gene locus

Kaposi's sarcoma-associated herpesvirus ORF30-33 locus encodes four genes with unknown functions. We performed transcriptional mapping of these genes. Northern-hybridization, 5'- and 3'-rapid amplification of cDNA ends, and DNA sequencing identified four transcripts of 3.7, 3.6, 2.7, and 1.4 kb, none of which has alternative splicing. While all transcripts have the same termination site, their start sites vary. All transcripts are not expressed or only weakly expressed in latent cells but can be chemically induced. The 3.7 and 3.6 kb transcripts contain all four genes and are sensitive to cycloheximide (CH) but resistant to phosphonoacetic acid (PAA), indicating that they are early lytic transcripts. The 2.7 kb transcript contains ORF32 and ORF33 genes while the 1.4 kb transcript contains the ORF33 gene. Both transcripts are sensitive to CH and PAA, indicating that they are late lytic transcripts. Furthermore, we identified four promoters with functional TATA boxes, none of which is directly transactivated by RTA. Examination of the 5' untranslated region of ORF31 failed to identify any functional internal ribosome entry sites. These results define the transcriptional patterns of the ORF30-33 locus, which should help the delineation of its function.

Yin Yang-1 Regulates the Characterized Murine Focal Adhesion-Associated Protein Promoter

The focal adhesion-associated protein (FAAP), product of the murine D10Wsu52e gene, is involved in modulating cell adhesion dynamics. The ubiquitously expressed protein belongs to the highly conserved UPF0027 family, the newly identified RNA >p ligase family. To understand the mechanisms underlying FAAP expression and regulation, we first mapped its major transcription start site at the nucleotide 79 bp upstream of the ATG codon. The murine FAAP 2.1 kb 5'-flanking region was cloned, analyzed, and aligned with the corresponding 1.7 kb region of its human homolog HSPC117. Despite the differences in activity, cell in vitro transfection and testis in vivo electroporation identified a 0.2 kb efficient promoter region lacking a functional TATA-box. Gel shift assays confirmed the specific interaction between Yin Yang-1 (YY1) and the potential element in the proximal region of the FAAP promoter. Site mutation, truncation, RNAi, and overexpression analyses suggested that YY1 is an important regulator of the FAAP promoter.

Functional characterization of the human translocator protein (18 kDa) gene promoter in human breast cancer cell lines

The translocator protein (18 kDa; TSPO) is a mitochondrial drug- and cholesterol-binding protein that has been implicated in several processes, including steroidogenesis, cell proliferation, and apoptosis. Expression of the human TSPO gene is elevated in several cancers. To understand the molecular mechanisms that regulate TSPO expression in human breast cancer cells, the TSPO promoter was identified, cloned, and functionally characterized in poor-in-TSPO hormone-dependent, non-aggressive MCF-7 cells and rich-in-TSPO hormone-independent, aggressive, and metastatic MDA-MB-231 breast cancer cells. RNA ligase-mediated 5′-rapid amplification of cDNA ends analysis indicated transcription initiated at multiple sites downstream of a GC-rich promoter that lacks functional TATA and CCAAT boxes. Deletion analysis indicated that the region from − 121 to + 66, which contains five putative regulatory sites known as GC boxes, was sufficient to induce reporter activity up to 24-fold in MCF-7 and nearly 120-fold in MDA-MB-231 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1, Sp3 and Sp4 bind to these GC boxes in vitro and to the endogenous TSPO promoter. Silencing of Sp1, Sp3 and Sp4 gene expression reduced TSPO levels. In addition, TSPO expression was epigenetically regulated at one or more of the identified GC boxes. Disruption of the sequence downstream of the main start site of TSPO differentially regulated TSPO promoter activity in MCF-7 and MDA-MB-231 cells, indicating that essential elements contribute to its differential expression in these cells. Taken together, these experiments constitute the first in-depth functional analysis of the human TSPO gene promoter and its transcriptional regulation.

Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.

Identification and characterization of the promoter region of Kaposi's sarcoma-associated herpesvirus ORF11

Open reading frame 11 (ORF11) of Kaposi's sarcoma-associated herpesvirus belongs to a herpesviral homologous protein family that is conserved among members of the gamma-herpesvirus subfamily. Little is known about the function of ORF11 and how this viral gene is regulated in KSHV life cycle. In this study, we have characterized the major transcript of the ORF11 gene, which is located adjacent and in the opposite orientation to a well-characterized viral IL-6 gene. Northern blot analysis revealed that the ORF11 gene is lytic viral gene with delayed-early expression kinetics. We have determined the 5′ and 3′ untranslated region of the unspliced ORF11 transcript and identified both the transcription start site and the transcription termination site. Core promoter region, representing ORF11 promoter activity, was mapped to a 160 nt fragment 5′ most proximal to the transcription start site. A functional TATA box was identified in the core promoter region. We also found that the characterized ORF11 gene promoter region is not responsive to Rta, the KSHV lytic switch protein. Our data help to elucidate transcription regulation of the KSHV ORF11 gene and to understand the biology of ORF11 in KSHV life cycle.

Minimal components of the RNA polymerase II transcription apparatus determine the consensus TATA box

In Saccharomyces cerevisiae, multiple approaches have arrived at a consensus TATA box sequence of TATA(T/A)A(A/T)(A/G). TATA-binding protein (TBP) affinity alone does not determine TATA box function. To discover how a minimal set of factors required for basal and activated transcription contributed to the sequence requirements for a functional TATA box, we performed transcription reactions using highly purified proteins and CYC1 promoter TATA box mutants. The TATA box consensus sequence is a good predictor of promoter activity. However, several nonconsensus sequences are almost fully functional, indicating that mechanistic requirements are not the only selective pressure on the TATA box. We also found that the effect of a mutation at a certain position is often dependent on other bases within a particular TATA box. Although activators and coactivators strongly influence TBP recruitment and stability at promoters, neither Mediator, the activator Gal4-V16, nor TFIID specifically compensate for the low transcription levels of the weak TATA boxes. The addition of Mediator to purified transcription reactions did, however, increase the functional selectivity for certain consensus TATA sequences. Transcription in whole-cell extracts or in vivo with these TATA box mutants indicated that factors, other than those in our purified system, may help initiate transcription from weak TATA boxes.

TBP binding capacity of the TATA box is associated with specific structural properties: AFM study of the IL-2R alpha gene promoter

DNA is not only a nucleotide sequence which allows the binding of regulators but its intrinsic structural properties such as curvature and flexibility are also viewed as playing an active role in the regulation of transcription. Our combination of computer modelling and AFM imaging allow direct access to DNA curvature and flexibility. We have searched for these DNA structural features involved in transcription regulation within the IL-2Rα gene promoter. Investigation of these structural characteristics shows concordant results. First, in the core promoter, the region containing the functional TATA box shows intrinsic curvature associated with a peculiar distribution of flexibility. Both these inherent properties are characteristic of this region as compared with the other parts of the promoter. Second, the proximal promoter exhibits two important regions: a first one flexible and curved, followed by a segment of rigid linear DNA, each localised within one of the two Positive Regulatory Regions PRRI and PRRII respectively. Based on these observations, we propose different roles for DNA curvature and/or flexibility in promoter sequences.

In silico identification of putative promoter motifs of White Spot Syndrome Virus

BackgroundWhite Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs.ResultsThe collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region.ConclusionThe data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

Identification and Characterization of a Functional TATA Box Polymorphism of the UDP Glucuronosyltransferase 1A7 Gene

UDP glucuronosyltransferases (UGT) detoxify bilirubin and therapeutic drugs, a process influenced by single nucleotide polymorphisms (SNPs) in their structural genes and promoter elements. UGT1A1*28 is a functional UGT promoter polymorphism associated with Gilbert's disease and severe irinotecan toxicity, which also occurs in the absence of UGT1A1*28. The aim of this study was to identify and characterize UGT promoter variants relevant for irinotecan detoxification. Recombinant UGT1A proteins were analyzed for irinotecan metabolite glucuronidation by UGT activity assays. In 427 healthy blood donors and 71 homozygous UGT1A1*28 carriers, the 5'-untranslated region of the UGT1A7 gene locus was studied. An SNP was detected by allelic discrimination and characterized by reporter gene experiments. A novel -57 T--> G SNP with a gene frequency of 0.39 in healthy blood donors was identified in the putative TATA box of the UGT1A7 gene, reducing promoter activity to 30%. It is in linkage dysequilibrium with a variant of the UGT1A7 first exon that is present in the reduced-activity UGT1A7*3 and UGT1A7*4 alleles. Homozygous UGT1A1*28 carriers simultaneously carried this variant in 97%. We identified a novel reduced-function TATA box SNP of the UGT1A7 gene that catalyzes irinotecan metabolite detoxification. Its association with variants of the UGT1A1 promoter and UGT1A7 gene may influence irinotecan metabolism. Our finding emphasizes the importance of combinations of structural and regulatory gene polymorphisms that may be useful as markers of drug toxicity.

Recombinant and native Plasmodium falciparum TATA-binding-protein binds to a specific TATA box element in promoter regions

RNA polymerase II promoters in Plasmodium spp., like in most eukaryotes, have a bipartite structure. However, the identification of a functional TATA box located within the Plasmodium spp. core promoters has been difficult, mainly because of its high A + T content. Only few putative trans-acting elements have been identified in the malaria parasite genome such as a gene orthologous to the TATA box binding protein (PfTBP). In this study, we demonstrate that PfTBP is part of the DNA–protein complexes formed in the kahrp and gbp- 130 gene promoter regions. Supershift and footprinting assays performed with a GST–PfTBP fusion protein showed that PfTBP associates with a consensus TATA box sequence located 81 base pairs upstream of the transcription start site in the kahrp promoter region and with a TATA box-like (TGTAA) sequence at position −186 of the gbp- 130 gene promoter region. Chromatin immunoprecipitation assays confirmed that native PfTBP is able to associate in vivo with both TATA box elements. This is the first study that reports the identification of cis-acting sequences (TATAA and TGTAA) and their corresponding trans-acting (PfTBP) factor in P. falciparum.

A conserved initiator element on the mammalian poly(ADP-ribose) polymerase-1 promoters, in combination with flanking core elements, is necessary to obtain high transcriptional activity

Poly(ADP-ribose) polymerase-1 (PARP-1) is a conserved nuclear protein present in nearly all eukaryotes. In mammalian cells, its abundant expression and its ability to specifically bind to DNA strand breaks make it an important enzyme in the rapid cellular response to DNA damage. Although the promoter regions of the three known mammalian PARP-1 genes, from human, rat and mouse, are different, they share common features, such as multiple GC-rich regions, lack of a functional TATA box, and presence of a putative initiator element. In this study, we analyzed the core promoter region of the rat PARP-1 gene, and show that it contains a functional initiator element surrounding the transcription start site. This core element lies within a ∼40-base-pair region that is highly conserved in all three mammalian PARP-1 promoters. Furthermore, we show that other core elements located upstream and downstream of the PARP-1 initiator, including a functional Sp1 target site, synergize to regulate rat PARP-1 transcription. As the initiator region of all three PARP-1 gene promoters is highly conserved, their transcriptional regulation is likely achieved through similar mechanisms.

Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression: ITS POSITIVE INVOLVEMENT IN TRANSFORMING GROWTH FACTOR-β SIGNALING AND INHIBITION IN HYPOXIA

UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.