Publications by authors named "Tamar Juven-Gershon"

23 Publications

  • Page 1 of 1

Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach.

Biol Proced Online 2020 14;22:14. Epub 2020 Jul 14.

Department of Developmental Biology and Cancer Research, Faculty of Medicine, Institute for Medical Research Israel-Canada, Hebrew University, Jerusalem, Israel.

Background: The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags.

Results: We successfully applied the CRISPR-based co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo importance of the respective mutations. For the () gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the () gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing embryo.

Conclusions: The described approach has facilitated the successful generation of point mutations in two different chromosomes, by two different labs. Distinct phenotypes associated with the newly-generated genotype were identified, thus exemplifying the importance of investigating the in vivo role of specific nucleotides. In addition, detailed guidelines, recommendations and crossing schemes are provided in order to support the generation of additional genetically modified animals by the scientific community.
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http://dx.doi.org/10.1186/s12575-020-00123-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362497PMC
July 2020

Quantitative Analysis of Differential Expression of HOX Genes in Multiple Cancers.

Cancers (Basel) 2020 Jun 14;12(6). Epub 2020 Jun 14.

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.

Transcription factors encoded by () genes play numerous key functions during early embryonic development and differentiation. Multiple reports have shown that mis-regulation of gene expression plays key roles in the development of cancers. Their expression levels in cancers tend to differ based on tissue and tumor type. Here, we performed a comprehensive analysis comparing gene expression in different cancer types, obtained from The Cancer Genome Atlas (TCGA), with matched healthy tissues, obtained from Genotype-Tissue Expression (GTEx). We identified and quantified differential expression patterns that confirmed previously identified expression changes and highlighted new differential expression signatures. We discovered differential expression patterns that are in line with patient survival data. This comprehensive and quantitative analysis provides a global picture of genes' differential expression patterns in different cancer types.
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http://dx.doi.org/10.3390/cancers12061572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352544PMC
June 2020

Integration of multiple epigenomic marks improves prediction of variant impact in saturation mutagenesis reporter assay.

Hum Mutat 2019 09 23;40(9):1280-1291. Epub 2019 Jun 23.

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.

The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.
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http://dx.doi.org/10.1002/humu.23797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879779PMC
September 2019

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

PLoS One 2019 18;14(4):e0215695. Epub 2019 Apr 18.

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215695PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472829PMC
January 2020

Functional Screening of Core Promoter Activity.

Methods Mol Biol 2017 ;1651:77-91

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Life Sciences Building (212) Room 102, Ramat-Gan, Israel.

The core promoter is the DNA sequence that recruits the basal transcription machinery and directs accurate initiation of transcription. It is an active contributor to gene expression that can be rationally designed to manipulate the levels of expression. Core promoter function can be analyzed using different experimental approaches. Here, we describe the qualitative and quantitative analysis of engineered core promoter functions using the EGFP reporter gene that is driven by distinct core promoters. Expression plasmids are transfected into different mammalian cell lines, and the resulting fluorescence is monitored by live cell imaging , as well as by flow cytometry. In order to verify that the transcriptional activity of the examined core promoters is indeed a function of their activity, as opposed to differences in DNA uptake, real-time quantitative PCR analysis is performed. Importantly, the described methodology for functional screening of core promoter activity has enabled the analysis of engineered potent core promoters for extended time periods.
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http://dx.doi.org/10.1007/978-1-4939-7223-4_7DOI Listing
May 2018

Structural and Dynamics Characterization of the MerR Family Metalloregulator CueR in its Repression and Activation States.

Structure 2017 07 1;25(7):988-996.e3. Epub 2017 Jun 1.

The Chemistry Department, Faculty of Exact Sciences, Bar Ilan University, Ramat-Gan 5290002, Israel. Electronic address:

CueR (Cu export regulator) is a metalloregulator protein that "senses" Cu(I) ions with very high affinity, thereby stimulating DNA binding and the transcription activation of two other metalloregulator proteins. The crystal structures of CueR when unbound or bound to DNA and a metal ion are very similar to each other, and the role of CueR and Cu(I) in initiating the transcription has not been fully understood yet. Using double electron-electron resonance (DEER) measurements and structure modeling, we investigate the conformational changes that CueR undergoes upon binding Cu(I) and DNA in solution. We observe three distinct conformations, corresponding to apo-CueR, DNA-bound CueR in the absence of Cu(I) (the "repression" state), and CueR-Cu(I)-DNA (the "activation" state). We propose a detailed structural mechanism underlying CueR's regulation of the transcription process. The mechanism explicitly shows the dependence of CueR activity on copper, thereby revealing the important negative feedback mechanism essential for regulating the intracellular copper concentration.
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http://dx.doi.org/10.1016/j.str.2017.05.004DOI Listing
July 2017

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

Sci Rep 2016 09 15;6:33351. Epub 2016 Sep 15.

Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, 5290002, Israel.

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.
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http://dx.doi.org/10.1038/srep33351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024299PMC
September 2016

Engineered Promoters for Potent Transient Overexpression.

PLoS One 2016 12;11(2):e0148918. Epub 2016 Feb 12.

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148918PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752495PMC
July 2016

ElemeNT: a computational tool for detecting core promoter elements.

Transcription 2015 ;6(3):41-50

a The Mina and Everard Goodman Faculty of Life Sciences ; Bar-Ilan University ; Ramat Gan , Israel.

Core promoter elements play a pivotal role in the transcriptional output, yet they are often detected manually within sequences of interest. Here, we present 2 contributions to the detection and curation of core promoter elements within given sequences. First, the Elements Navigation Tool (ElemeNT) is a user-friendly web-based, interactive tool for prediction and display of putative core promoter elements and their biologically-relevant combinations. Second, the CORE database summarizes ElemeNT-predicted core promoter elements near CAGE and RNA-seq-defined Drosophila melanogaster transcription start sites (TSSs). ElemeNT's predictions are based on biologically-functional core promoter elements, and can be used to infer core promoter compositions. ElemeNT does not assume prior knowledge of the actual TSS position, and can therefore assist in annotation of any given sequence. These resources, freely accessible at http://lifefaculty.biu.ac.il/gershon-tamar/index.php/resources, facilitate the identification of core promoter elements as active contributors to gene expression.
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http://dx.doi.org/10.1080/21541264.2015.1067286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4581360PMC
May 2016

Structure-Function Analysis of the Drosophila melanogaster Caudal Transcription Factor Provides Insights into Core Promoter-preferential Activation.

J Biol Chem 2015 Jul 26;290(28):17293-305. Epub 2015 May 26.

From The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel and

Regulation of RNA polymerase II transcription is critical for the proper development, differentiation, and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator, and downstream core promoter element (DPE), which confer specific properties to the core promoter. We have previously discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. Here, we show that the mouse Caudal-related homeobox (Cdx) proteins (mCdx1, mCdx2, and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, we performed structure-function analysis. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) we discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz) Caudal target gene. Furthermore, the region containing both the homeodomain and the C terminus of Caudal was sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, we discovered that Drosophila CREB-binding protein (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP conferred the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that were unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner.
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http://dx.doi.org/10.1074/jbc.M114.632109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498068PMC
July 2015

The core promoter: At the heart of gene expression.

Biochim Biophys Acta 2015 Aug 28;1849(8):1116-31. Epub 2015 Apr 28.

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel. Electronic address:

The identities of different cells and tissues in multicellular organisms are determined by tightly controlled transcriptional programs that enable accurate gene expression. The mechanisms that regulate gene expression comprise diverse multiplayer molecular circuits of multiple dedicated components. The RNA polymerase II (Pol II) core promoter establishes the center of this spatiotemporally orchestrated molecular machine. Here, we discuss transcription initiation, diversity in core promoter composition, interactions of the basal transcription machinery with the core promoter, enhancer-promoter specificity, core promoter-preferential activation, enhancer RNAs, Pol II pausing, transcription termination, Pol II recycling and translation. We further discuss recent findings indicating that promoters and enhancers share similar features and may not substantially differ from each other, as previously assumed. Taken together, we review a broad spectrum of studies that highlight the importance of the core promoter and its pivotal role in the regulation of metazoan gene expression and suggest future research directions and challenges.
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http://dx.doi.org/10.1016/j.bbagrm.2015.04.003DOI Listing
August 2015

TRF2: TRansForming the view of general transcription factors.

Transcription 2015 14;6(1):1-6. Epub 2015 Jan 14.

a The Mina and Everard Goodman Faculty of Life Sciences , Bar-Ilan University , Ramat Gan , 5290002 , Israel.

Transcriptional regulation is pivotal for development and differentiation of organisms. Transcription of eukaryotic protein-coding genes by RNA polymerase II (Pol II) initiates at the core promoter. Core promoters, which encompass the transcription start site, may contain functional core promoter elements, such as the TATA box, initiator, TCT and downstream core promoter element. TRF2 (TATA-box-binding protein-related factor 2) does not bind TATA box-containing promoters. Rather, it is recruited to core promoters via sequences other than the TATA box. We review the recent findings implicating TRF2 as a basal transcription factor in the regulation of diverse biological processes and specialized transcriptional programs.
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http://dx.doi.org/10.1080/21541264.2015.1004980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422821PMC
January 2016

The core promoter composition establishes a new dimension in developmental gene networks.

Nucleus 2014 Jul-Aug;5(4):298-303

a The Mina and Everard Goodman Faculty of Life Sciences; Bar-Ilan University; Ramat Gan, Israel.

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II, which initiates transcription at the core promoter. The dorsal-ventral gene regulatory network (GRN) includes multiple genes that are activated by different nuclear concentrations of the Dorsal transcription factor along the dorsal-ventral axis. Downstream core promoter element (DPE)-containing genes are conserved and highly prevalent among Dorsal target genes. Moreover, the DPE motif is functional in multiple Dorsal target genes, as mutation of the DPE results in the loss of transcriptional activity. Furthermore, analysis of hybrid enhancer-promoter constructs reveals that the core promoter composition plays a pivotal role in the transcriptional output. Importantly, we provide in vivo evidence that expression driven by the homeotic Antennapedia P2 promoter during Drosophila embryogenesis is dependent on the DPE. Taken together, we propose that transcriptional regulation results from the interplay between enhancers and core promoter composition, thus establishing a novel dimension in developmental GRNs.
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http://dx.doi.org/10.4161/nucl.29838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4152343PMC
September 2015

The FOXO transcription factor DAF-16 bypasses ire-1 requirement to promote endoplasmic reticulum homeostasis.

Cell Metab 2014 Nov;20(5):870-881

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 5290002 Ramat Gan, Israel. Electronic address:

The unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional.We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis.
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http://dx.doi.org/10.1016/j.cmet.2014.09.006DOI Listing
November 2014

Drosophila TRF2 is a preferential core promoter regulator.

Genes Dev 2014 Oct 15;28(19):2163-74. Epub 2014 Sep 15.

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated genes. Using primer extension analysis and reporter assays, we show the importance of the DPE in transcriptional regulation of TRF2 target genes. It was previously shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, we discovered that short TRF2-bound DNA oligos are enriched for Inr and DPE motifs. Taken together, our findings highlight the role of short TRF2 as a preferential core promoter regulator.
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http://dx.doi.org/10.1101/gad.245670.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180977PMC
October 2014

Core promoter functions in the regulation of gene expression of Drosophila dorsal target genes.

J Biol Chem 2014 Apr 14;289(17):11993-12004. Epub 2014 Mar 14.

Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. Electronic address:

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes.
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http://dx.doi.org/10.1074/jbc.M114.550251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002106PMC
April 2014

Human TFIID binds to core promoter DNA in a reorganized structural state.

Cell 2013 Jan;152(1-2):120-31

Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA.

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.
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http://dx.doi.org/10.1016/j.cell.2012.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552382PMC
January 2013

Regulation of gene expression via the core promoter and the basal transcriptional machinery.

Dev Biol 2010 Mar 13;339(2):225-9. Epub 2009 Aug 13.

Section of Molecular Biology, 0347, University of California, San Diego, La Jolla, CA 92093-0347, USA.

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional regulatory element. There are two main strategies for transcription initiation - focused and dispersed initiation. In focused initiation, transcription starts from a single nucleotide or within a cluster of several nucleotides, whereas in dispersed initiation, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides. Focused initiation is the predominant means of transcription in simpler organisms, whereas dispersed initiation is observed in approximately two-thirds of vertebrate genes. Regulated genes tend to have focused promoters, and constitutive genes typically have dispersed promoters. Hence, in vertebrates, focused promoters are used in a small but biologically important fraction of genes. The properties of focused core promoters are dependent upon the presence or absence of sequence motifs such as the TATA box and DPE. For example, Caudal, a key regulator of the homeotic gene network, preferentially activates transcription from DPE- versus TATA-dependent promoters. The basal transcription factors, which act in conjunction with the core promoter, are another important component in the regulation of gene expression. For instance, upon differentiation of myoblasts to myotubes, the cells undergo a switch from a TFIID-based transcription system to a TRF3-TAF3-based system. These findings suggest that the core promoter and basal transcription factors are important yet mostly unexplored components in the regulation of gene expression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830304PMC
http://dx.doi.org/10.1016/j.ydbio.2009.08.009DOI Listing
March 2010

Caudal, a key developmental regulator, is a DPE-specific transcriptional factor.

Genes Dev 2008 Oct;22(20):2823-30

Section of Molecular Biology, University of California at San Diego, La Jolla, California 92093, USA.

The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promoter element (DPE). To understand the basis for core promoter diversity, we explored potential biological functions of the DPE. We found that nearly all of the Drosophila homeotic (Hox) gene promoters, which lack TATA-box elements, contain functionally important DPE motifs that are conserved from Drosophila melanogaster to Drosophila virilis. We then discovered that Caudal, a sequence-specific transcription factor and key regulator of the Hox gene network, activates transcription with a distinct preference for the DPE relative to the TATA box. The specificity of Caudal activation for the DPE is particularly striking when a BRE(u) core promoter motif is associated with the TATA box. These findings show that Caudal is a DPE-specific activator and exemplify how core promoter diversity can be used to establish complex regulatory networks.
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http://dx.doi.org/10.1101/gad.1698108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2569877PMC
October 2008

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription.

Genes Dev 2008 Sep 14;22(17):2353-8. Epub 2008 Aug 14.

Section of Molecular Biology, University of California at San Diego, La Jolla, California 92093, USA.

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional module. RNAi depletion and overexpression experiments revealed a genetic circuit that controls the balance of transcription from two core promoter motifs, the TATA box and the downstream core promoter element (DPE). In this circuit, TBP activates TATA-dependent transcription and represses DPE-dependent transcription, whereas Mot1 and NC2 block TBP function and thus repress TATA-dependent transcription and activate DPE-dependent transcription. This regulatory circuit is likely to be one means by which biological networks can transmit transcriptional signals, such as those from DPE-specific and TATA-specific enhancers, via distinct pathways.
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http://dx.doi.org/10.1101/gad.1681808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532932PMC
September 2008

The RNA polymerase II core promoter - the gateway to transcription.

Curr Opin Cell Biol 2008 Jun 22;20(3):253-9. Epub 2008 Apr 22.

Section of Molecular Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0347, USA.

The RNA polymerase II core promoter is generally defined to be the sequence that directs the initiation of transcription. This simple definition belies a diverse and complex transcriptional module. There are two major types of core promoters - focused and dispersed. Focused promoters contain either a single transcription start site or a distinct cluster of start sites over several nucleotides, whereas dispersed promoters contain several start sites over 50-100 nucleotides and are typically found in CpG islands in vertebrates. Focused promoters are more ancient and widespread throughout nature than dispersed promoters; however, in vertebrates, dispersed promoters are more common than focused promoters. In addition, core promoters may contain many different sequence motifs, such as the TATA box, BRE, Inr, MTE, DPE, DCE, and XCPE1, that specify different mechanisms of transcription and responses to enhancers. Thus, the core promoter is a sophisticated gateway to transcription that determines which signals will lead to transcription initiation.
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http://dx.doi.org/10.1016/j.ceb.2008.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2586601PMC
June 2008

Rational design of a super core promoter that enhances gene expression.

Nat Methods 2006 Nov;3(11):917-22

Section of Molecular Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

Transcription is a critical component in the expression of genes. Here we describe the design and analysis of a potent core promoter, termed super core promoter 1 (SCP1), which directs high amounts of transcription by RNA polymerase II in metazoans. SCP1 contains four core promoter motifs-the TATA box, initiator (Inr), motif ten element (MTE) and downstream promoter element (DPE)-in a single promoter, and is distinctly stronger than the cytomegalovirus (CMV) IE1 and adenovirus major late (AdML) core promoters both in vitro and in vivo. Each of the four core promoter motifs is needed for full SCP1 activity. SCP1 is bound efficiently by TFIID and exhibits a high propensity to form productive transcription complexes. SCP1 and related super core promoters (SCPs) with multiple core promoter motifs will be useful for the biophysical analysis of TFIID binding to DNA, the biochemical investigation of the transcription process and the enhancement of gene expression in cells.
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http://dx.doi.org/10.1038/nmeth937DOI Listing
November 2006