Figure 1 Theory Meets Figure 2 Experiments in the Study of Gene Expression

It is tempting to believe that we now own the genome. The ability to read and rewrite it at will has ushered in a stunning period in the history of science. Nonetheless, there is an Achilles’ heel exposed by all of the genomic data that has accrued: We still do not know how to interpret them. Many genes are subject to sophisticated programs of transcriptional regulation, mediated by DNA sequences that harbor binding sites for transcription factors, which can up- or down-regulate gene expression depending upon environmental conditions. This gives rise to an input–output function describing how the level of expression depends upon the parameters of the regulated gene—for instance, on the number and type of binding sites in its regulatory sequence. In recent years, the ability to make precision measurements of expression, coupled with the ability to make increasingly sophisticated theoretical predictions, has enabled an explicit dialogue between theory and experiment that holds the promise of covering this genomic Achilles’ heel. The goal is to reach a predictive understanding of transcriptional regulation that makes it possible to calculate gene expression levels from DNA regulatory sequence. This review focuses on the canonical simple repression motif to ask how well the models that have been used to characterize it actually work. We consider a hierarchy of increasingly sophisticated experiments in which the minimal parameter set learned at one level is applied to make quantitative predictions at the next. We show that these careful quantitative dissections provide a template for a predictive understanding of the many more complex regulatory arrangements found across all domains of life.

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Novel Mechanism of Positive versus Negative Regulation by Thyroid Hormone Receptor β1 (TRβ1) Identified by Genome-wide Profiling of Binding Sites in Mouse Liver

Journal of Biological Chemistry ◽

10.1074/jbc.m113.521450 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1313-1328 ◽

Cited By ~ 69

Author(s):

Preeti Ramadoss ◽

Brian J. Abraham ◽

Linus Tsai ◽

Yiming Zhou ◽

Ricardo H. Costa-e-Sousa ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Thyroid Hormone ◽

Binding Sites ◽

Hormone Receptor ◽

Target Genes ◽

Thyroid Hormone Receptor ◽

Negative Regulation ◽

Genome Wide

Triiodothyronine (T3) regulates key metabolic processes in the liver through the thyroid hormone receptor, TRβ1. However, the number of known target genes directly regulated by TRβ1 is limited, and the mechanisms by which positive and especially negative transcriptional regulation occur are not well understood. To characterize the TRβ1 cistrome in vivo, we expressed a biotinylated TRβ1 in hypo- and hyperthyroid mouse livers, used ChIP-seq to identify genomic TRβ1 targets, and correlated these data with gene expression changes. As with other nuclear receptors, the majority of TRβ1 binding sites were not in proximal promoters but in the gene body of known genes. Remarkably, T3 can dictate changes in TRβ1 binding, with strong correlation to T3-induced gene expression changes, suggesting that differential TRβ1 binding regulates transcriptional outcome. Additionally, DR-4 and DR-0 motifs were significantly enriched at binding sites where T3 induced an increase or decrease in TRβ1 binding, respectively, leading to either positive or negative regulation by T3. Taken together, the results of this study provide new insights into the mechanisms of transcriptional regulation by TRβ1 in vivo.

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Transcriptional Regulation in the G1-S Cell Cycle Stage in Fungi: Insights through Computational Analysis

The Open Bioinformatics Journal ◽

10.2174/1875036201206010043 ◽

2012 ◽

Vol 6 (1) ◽

pp. 43-54

Author(s):

Viktor Martyanov ◽

Robert H. Gross

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Transcriptional Regulation ◽

Transcription Factors ◽

Dna Sequences ◽

Binding Sites ◽

Positional Distribution ◽

Upstream Sequence ◽

Regulatory Pathways ◽

Binding Factor

The transcription factor complexes Mlu1-box binding factor (MBF) and Swi4/6 cell cycle box binding factor (SBF) regulate the cell cycle in Saccharomyces cerevisiae. They activate hundreds of genes and are responsible for nor-mal cell cycle progression from G1 to S phase. We investigated the conservation of MBF and SBF binding sites during fungal evolution. Orthologs of S. cerevisiae targets of these transcription factors were identified in 37 fungal species and their upstream regions were analyzed for putative transcription factor binding sites. Both groups displayed enrichment in specific putative regulatory DNA sequences in their upstream regions and showed different preferred upstream motif loca-tions, variable patterns of evolutionary conservation of the motifs and enrichment in unique biological functions for the regulated genes. The results indicate that despite high sequence similarity of upstream DNA motifs putatively associated with G1-S transcriptional regulation by MBF and SBF transcription factors, there are important upstream sequence feature differences that may help differentiate the two seemingly similar regulatory modes. The incorporation of upstream motif sequence comparison, positional distribution and evolutionary variability of the motif can complement functional infor-mation about roles of the respective gene products and help elucidate transcriptional regulatory pathways and functions.

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A Global View of Transcriptional Regulation by Nuclear Receptors: Gene Expression, Factor Localization, and DNA Sequence Analysis

Nuclear Receptor Signaling ◽

10.1621/nrs.06005 ◽

2008 ◽

Vol 6 (1) ◽

pp. nrs.06005 ◽

Cited By ~ 52

Author(s):

Miltiadis Kininis ◽

W. Lee Kraus

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Rna Polymerase Ii ◽

Nuclear Receptors ◽

Dna Sequences ◽

Target Genes ◽

Large Body ◽

Pol Ii ◽

Global View ◽

Genomic Analyses

Recent genomic analyses of transcription factor binding, histone modification, and gene expression have provided a global view of transcriptional regulation by nuclear receptors (NRs) that complements an existing large body of literature on gene-specific studies. The picture emerging from these genomic studies indicates that NRs bind at promoter-proximal and promoter-distal enhancers in conjunction with other transcription factors (e.g., activator protein-1, Sp1 and FOXA1). This binding promotes the recruitment of coregulators that mediate the posttranslational modification of histones at promoters and enhancers. Ultimately, signaling through liganded NRs stimulates changes in the occupancy of RNA polymerase II (Pol II) or the activation of preloaded Pol II at target promoters. Chromosomal looping and/or Pol II tracking may underlie promoter-enhancer communication. Interestingly, the direct target genes of NR signaling represent a limited subset of all the genes regulated by NR ligands, with the rest being regulated through secondary effects. As suggested by previous gene-specific analyses, NR-mediated outcomes are highly cell type- and promoter-specific, highlighting the complexity of transcriptional regulation by NRs and the value of genomic analyses for identifying commonly shared patterns. Overall, NRs share common themes in their patterns of localization and transcriptional regulation across mammalian genomes. In this review, we provide an overview of recent advances in the understanding of NR-mediated transcription garnered from genomic analyses of gene expression, factor localization, and target DNA sequences.

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Role of polycomb repressive complex 2 in regulation of human transcription factor gene expression

10.1101/2021.08.04.455052 ◽

2021 ◽

Author(s):

Jay Brown

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Binding Sites ◽

Regulatory Control ◽

Polycomb Repressive Complex ◽

Control Of Gene Expression ◽

Polycomb Repressive Complex 2

Control of gene expression is now recognized as a central issue in the field of molecular biology. We now know the sequences of many genomes including that of the human genome, and we know the nature of many pathways involved in control of gene expression. It remains difficult, however, to look at the DNA sequences surrounding a particular gene and tell which methods of regulatory control are in use. I have been pursuing the idea that progress might be made by comparing the regulatory regions of paired gene populations in which one population is strongly expressed and the other weakly. Here I report the results obtained with human genes encoding transcription factors (TF). In this population, broadly expressed genes are strongly expressed while tissue targeted TF expression is suppressed in most tissues. The results demonstrated that the promoter region of broadly expressed TF genes is enriched in binding sites for POLR2A, a component of RNA polymerase II while promoters of tissue targeted genes are enriched in EZH2, a subunit of polycomb repressive complex 2 (PRC2). It was rare to observe promoters with binding sites for both POLR2A and EZH2. The findings are interpreted to indicate that strong expression of broadly expressed TF genes is due to the presence of RNA polymerase II at the promoter while weak expression of tissue targeted promoters results from the presence of PRC2. Finally, transcription factor families were compared in the proportion of broadly expressed and tissue targeted genes they contain. The results demonstrated that most families possess both broadly expressed and tissue targeted members. For instance, this was the case with 16 of 20 TF families examined. The results are interpreted to indicate that while individual TFs such as EZH2 may be specific for broadly expressed or tissue targeted genes, this is not a property of most TF families.

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Characterization of the rat vasoactive intestinal polypeptide receptor gene 5′ region

Biochemical Journal ◽

10.1042/bj3080719 ◽

1995 ◽

Vol 308 (3) ◽

pp. 719-723 ◽

Cited By ~ 16

Author(s):

L Pei ◽

S Melmed

Keyword(s):

Gene Expression ◽

Vasoactive Intestinal Polypeptide ◽

Dna Sequences ◽

Binding Sites ◽

Receptor Gene ◽

Rat Lung ◽

Rnase Protection ◽

Receptor Gene Expression ◽

Intestinal Polypeptide ◽

Vip Receptor

The broad spectrum of vasoactive intestinal polypeptide (VIP) cellular functions are mediated by high-affinity binding sites. To determine regulation of the VIP receptor gene expression, we have isolated and characterized two genomic clones that contain the first three exons and the 5′ flanking region of the VIP receptor gene. Using RNase protection assays, receptor gene expression was detected in adult rat lung, liver and intestine, but not in fetal lung, indicating that VIP receptor is expressed in diverse tissues, and its expression is differentially regulated during lung development. The transcription start site of the gene was mapped to a cytosine residue, 76 bp upstream from the ATG initiation codon. Transfection into rat lung cell lines shows that although 126 bp of the VIP receptor 5′ DNA sequences are capable of activating VIP receptor gene basal transcription 30-fold over a promoterless control, 488 bp of the 5′ sequences further induce this activation to 97-fold over control. However, inclusion of up to 859 bp 5′ sequences results in a decrease in basal promoter activity (31-fold over control), indicating that while sequences between -126 and -488 bp contain potential enhancer sequences, sequences between -488 and -859 bp may include a transcriptional repressor sequence. Deletion analysis shows that transcription factor Sp1 plays an important role in activating basal promoter of the VIP receptor gene. DNase I footprinting and gel-mobility-shift assays show that Sp1 binds to its consensus binding sites in the VIP receptor promoter, suggesting that interaction of Sp1 with VIP receptor promoter transactivates this gene.

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Discovery of Over-Represented Words in Intron 1s of Drosophila Ribosomal Protein Genes

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.707.117 ◽

2014 ◽

Vol 707 ◽

pp. 117-120

Author(s):

Hui Min Li ◽

Zhi Gang Yang ◽

Dan Chen

Keyword(s):

Transcriptional Regulation ◽

Ribosomal Protein ◽

Dna Sequences ◽

Binding Sites ◽

Regulatory Elements ◽

Score Statistic ◽

Z Score ◽

Biological Functions ◽

Ribosomal Protein Genes

Most of studies on transcriptional regulation mainly focus on upstream regions of genes. More and more recent researches indicate that introns may have important biological functions in transcription regulation of genes. The characterization of words in DNA sequences can be facilitated by the sequences’ functions. Using U-score and Z-score statistic, respectively, we extracted some over-represented words in intron 1s of ribosomal protein genes. A majority of them are accordance with known transcriptional factor binding sites and are potential regulatory elements. And, the detected over-represented words are more likely to form wider potential sequences and are denser in intron 1s of RP genes. We speculate the properties of these words may be associated with the transcriptional regulation of RP genes.

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The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-κB

Scientific Reports ◽

10.1038/s41598-021-82735-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hee-Eun Lee ◽

Sang-Je Park ◽

Jae-Won Huh ◽

Hiroo Imai ◽

Heui-Soo Kim

Keyword(s):

Gene Expression ◽

Transposable Elements ◽

Human Genome ◽

Dna Sequences ◽

Binding Sites ◽

Luciferase Assay ◽

Enhancer Activity ◽

Long Interspersed Nuclear Elements ◽

Long Interspersed Nuclear Element ◽

Affect Gene Expression

AbstractTransposable elements (TEs) are DNA sequences that cut or introduced into the genome, and they represent a massive portion of the human genome. TEs generate a considerable number of microRNAs (miRNAs) are derived from TEs (MDTEs). Numerous miRNAs are related to cancer, and hsa-miRNA-625 is a well-known oncomiR derived from long interspersed nuclear elements (LINEs). The relative expression of hsa-miRNA-625-5p differs in humans, chimpanzees, crab-eating monkeys, and mice, and four primers were designed against the 3′UTR of GATAD2B to analyze the different quantities of canonical binding sites and the location of miRNA binding sites. Luciferase assay was performed to score for the interaction between hsa-miRNA-625 and the 3′UTR of GATAD2B, while blocking NF-κB. In summary, the different numbers of canonical binding sites and the locations of miRNA binding sites affect gene expression, and NF-κB induces the enhancer activity of hsa-miRNA-625-5p by sharing the binding sites.

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Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites

Nucleic Acids Research ◽

10.1093/nar/gkaa1234 ◽

2020 ◽

Author(s):

Tiebin Wang ◽

Nathan Tague ◽

Stephen A Whelan ◽

Mary J Dunlop

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Metabolic Engineering ◽

Dna Sequences ◽

Binding Sites ◽

Metabolic Flux ◽

Fold Increase ◽

Arginine Biosynthesis ◽

Knock Out ◽

Transcription Factor Decoy

Abstract Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering.

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The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant

10.1101/2020.11.18.389122 ◽

2020 ◽

Author(s):

Catherine A. Klancher ◽

George Minasov ◽

Ram Podicheti ◽

Douglas B. Rusch ◽

Triana N. Dalia ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Vibrio Cholerae ◽

Dna Sequences ◽

Binding Domain ◽

Structural Basis ◽

Striking Difference ◽

Dna Binding Domain ◽

Structural Domains ◽

Domains Of Life

AbstractSequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight “turn” of the HTH is replaced with an extended loop containing a β-sheet, a variant which we term the “helix-sheet-helix”. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.ImportanceRegulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

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Ash1 and Tup1 Dependent Repression of the Saccharomyces cerevisiae HO promoter Requires Activator-Dependent Nucleosome Eviction

10.1101/2020.09.29.318063 ◽

2020 ◽

Author(s):

Emily J. Parnell ◽

Timothy J. Parnell ◽

Chao Yan ◽

Lu Bai ◽

David J. Stillman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Transcription Factor ◽

Transcriptional Regulation ◽

Dna Sequences ◽

Binding Sites ◽

Genome Wide ◽

Intergenic Regions ◽

Mother Cells ◽

Activation Cycle

ABSTRACTTranscriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

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