From promoter motif to cardiac function: A single DPE motif affects transcription regulation and organ function in vivo.

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7bp substitution mutation results in massive perturbation of the Tinman regulatory network orchestrating dorsal musculature, manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.

ElemeNT 2023: an enhanced tool for detection and curation of core promoter elements.

MOTIVATION: Prediction and identification of core promoter elements and transcription factor binding sites is essential for understanding the mechanism of transcription initiation and deciphering the biological activity of a specific locus. Thus, there is a need for an up-to-date tool to detect and curate core promoter elements/motifs in any provided nucleotide sequences. RESULTS: Here, we introduce ElemeNT 2023-a new and enhanced version of the Elements Navigation Tool, which provides novel capabilities for assessing evolutionary conservation and for readily evaluating the quality of high-throughput transcription start site (TSS) datasets, leveraging preferential motif positioning. ElemeNT 2023 is accessible both as a fast web-based tool and via command line (no coding skills are required to run the tool). While this tool is focused on core promoter elements, it can also be used for searching any user-defined motif, including sequence-specific DNA binding sites. Furthermore, ElemeNT's CORE database, which contains predicted core promoter elements around annotated TSSs, is now expanded to cover 10 species, ranging from worms to human. In this applications note, we describe the new workflow and demonstrate a case study using ElemeNT 2023 for core promoter composition analysis of diverse species, revealing motif prevalence and highlighting evolutionary insights. We discuss how this tool facilitates the exploration of uncharted transcriptomic data, appraises TSS quality, and aids in designing synthetic promoters for gene expression optimization. Taken together, ElemeNT 2023 empowers researchers with comprehensive tools for meticulous analysis of sequence elements and gene expression strategies. AVAILABILITY AND IMPLEMENTATION: ElemeNT 2023 is freely available at https://www.juven-gershonlab.org/resources/element-v2023/. The source code and command line version of ElemeNT 2023 are available at https://github.com/OritAdato/ElemeNT. No coding skills are required to run the tool.

A single DPE core promoter motif contributes to in vivo transcriptional regulation and affects cardiac function.

Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we show that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. We demonstrate the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.

Rapid Biosensing Method for Detecting Protein-DNA Interactions.

Identifying and investigating protein-DNA interactions, which play significant roles in many biological processes, is essential for basic and clinical research. Current techniques for identification of protein-DNA interactions are laborious, time-consuming, and suffer from nonspecific binding and limited sensitivity. To overcome these challenges and assess protein-DNA interactions, we use a magnetic modulation biosensing (MMB) system. In MMB, one of the interacting elements (protein or DNA) is immobilized to magnetic beads, and the other is coupled to a fluorescent molecule. Thus, the link between the magnetic bead and the fluorescent molecule is established only when binding occurs, enabling detection of the protein-DNA interaction. Using magnetic forces, the beads are concentrated and manipulated in a periodic motion in and out of a laser beam, producing a detectable oscillating signal. Using MMB, we detected protein-DNA interactions between short GC-rich DNA sequences and both a purified specificity protein 1 (Sp1) and an overexpressed Buttonhead (BTD) protein in a cell lysate. The specificity of the interactions was assessed using mutated DNA sequences and competition experiments. The assays were experimentally compared with commonly used electrophoretic mobility shift assay, which takes approximately 4-72 h. In comparison, the MMB-based assay's turnaround time is ∼2 h, and it provides unambiguous results and quantitative measures of performance. The MMB system uses simple and cheap components, making it an attractive alternative method over current costly and time-consuming techniques for analyzing protein-DNA interactions. Therefore, we anticipate that the MMB-based technique will significantly advance the detection of protein-DNA interactions in biomedical research.

Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation and is altered with age.

Neural stem cells (NSCs) in the adult and aged brain are largely quiescent, and require transcriptional reprogramming to re-enter the cell cycle. However, the mechanisms underlying these changes and how they are altered with age remain undefined. Here, we identify the chromatin accessibility differences between primary neural stem/progenitor cells in quiescent and activated states. These distinct cellular states exhibit shared and unique chromatin profiles, both associated with gene regulation. Accessible chromatin states specific to activation or quiescence are active enhancers bound by key pro-neurogenic and quiescence factors. In contrast, shared sites are enriched for core promoter elements associated with translation and metabolism. Unexpectedly, through integrated analysis, we find that many sites that become accessible during NSC activation are linked to gene repression and associated with pro-quiescence factors, revealing a novel mechanism that may preserve quiescence re-entry. Furthermore, we report that in aged NSCs, chromatin regions associated with metabolic and transcriptional functions bound by key pro-quiescence transcription factors lose accessibility, suggesting a novel mechanism of age-associated NSC dysfunction. Together, our findings reveal how accessible chromatin states regulate the transcriptional switch between NSC quiescence and activation, and how this switch is affected with age.

The Core Promoter Is a Regulatory Hub for Developmental Gene Expression.

The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression.

Computational identification and experimental characterization of preferred downstream positions in human core promoters.

Metazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.

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

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 white co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified Drosophila melanogaster 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 Nucleoporin107 (Nup107) gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the tinman (tin) gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing Drosophila melanogaster 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.

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

Transcription factors encoded by Homeobox (HOX) genes play numerous key functions during early embryonic development and differentiation. Multiple reports have shown that mis-regulation of HOX 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 HOX 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 HOX genes' differential expression patterns in different cancer types.

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

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.

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

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.

Functional Screening of Core Promoter Activity.

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.

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

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.

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

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.

Engineered Promoters for Potent Transient Overexpression.

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.

ElemeNT: a computational tool for detecting core promoter elements.

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.

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

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.

The core promoter: At the heart of gene expression.

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.

TRF2: TRansForming the view of general transcription factors.

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.