scholarly journals Multiplexed dissection of a model human transcription factor binding site architecture

2019 ◽  
Author(s):  
Jessica E. Davis ◽  
Kimberly D. Insigne ◽  
Eric M. Jones ◽  
Quinn B Hastings ◽  
Sriram Kosuri
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Regulation Of Transcription ◽  
Genomic Context ◽  
Transcriptional Responses ◽  
Transcription Start Sites ◽  
Regulatory Architecture ◽  
The Impact

AbstractIn eukaryotes, transcription factors orchestrate gene expression by binding to TF-Binding Sites (TFBSs) and localizing transcriptional co-regulators and RNA Polymerase II to cis-regulatory elements. The strength and regulation of transcription can be modulated by a variety of factors including TFBS composition, TFBS affinity and number, distance between TFBSs, distance of TFBSs to transcription start sites, and epigenetic modifications. We still lack a basic comprehension of how such variables shaping cis-regulatory architecture culminate in quantitative transcriptional responses. Here we explored how such factors determine the transcriptional activity of a model transcription factor, the c-AMP Response Element (CRE) binding protein. We measured expression driven by 4,602 synthetic regulatory elements in a massively parallel reporter assay (MPRA) exploring the impact of CRE number, affinity, distance to the promoter, and spacing between multiple CREs. We found the number and affinity of CREs within regulatory elements largely determines overall expression, and this relationship is shaped by the proximity of each CRE to the downstream promoter. In addition, while we observed expression periodicity as the CRE distance to the promoter varied, the spacing between multiple CREs altered this periodicity. Finally, we compare library expression between an episomal MPRA and a new, genomically-integrated MPRA in which a single synthetic regulatory element is present per cell at a defined locus. We observe that these largely recapitulate each other although weaker, non-canonical CREs exhibited greater activity in the genomic context.

scholarly journals Euplotid: A quantized geometric model of the eukaryotic cell

2017 ◽  
Author(s):  
Diego Borges-Rivera
Keyword(s):  
Transcription Factor ◽  
Geometric Model ◽  
Building Blocks ◽  
Regulatory Elements ◽  
Eukaryotic Cell ◽  
Chromatin Accessibility ◽  
Regulatory Architecture ◽  
The Neural Network ◽  
Transition Mutation ◽  
The Impact

Life continues to shock and amaze us, reminding us that truth is far stranger than fiction. http://Euplotid.io is a quantized, geometric model of the eukaryotic cell, an attempt at quantifying the incredible complexity that gives rise to a living cell by beginning from the smallest unit, a quanta. Starting from the very bottom we are able to build the pieces which when hierarchically and combinatorially combined produce the emergent complex behavior that even a single celled organism can show. Euplotid is composed of a set of quantized geometric 3D building blocks and constantly evolving dockerized bioinformatic pipelines enabling a user to build and interact with the local regulatory architecture of every gene starting from DNA-interactions, chromatin accessibility, and RNA-sequencing. Reads are quantified using the latest computational tools and the results are normalized, quality-checked, and stored. The local regulatory architecture of each gene is built using a Louvain based graph partitioning algorithm parameterized by the chromatin extrusion model and CTCF-CTCF interactions. Cis-Regulatory Elements are defined using chromatin accessibility peaks which are mapped to Transcriptional Start Sites based on inclusion within the same neighborhood. Deep Neural Networks are trained in order to provide a statistical model mimicking transcription factor binding, giving the ability to identify all Transcription Factors within a given chromatin accessibility peak. By in-silico mutating and re-applying the neural network we are able to gauge the impact of a transition mutation on the binding of any transcription factor. The annotated output can be visualized in a variety of 1D, 2D, 3D and 4D ways overlaid with existing bodies of knowledge such as GWAS results or PDB structures. Once a particular CRE of interest has been identified a Base Editor mediated transition mutation can then be performed in a relevant model for further study.

scholarly journals Regulation of RNA Polymerase II Transcription Initiation and Elongation by Transcription Factor TFII-I

2021 ◽  
Vol 8 ◽  
Author(s):  
Niko Linzer ◽  
Alexis Trumbull ◽  
Rukiye Nar ◽  
Matthew D. Gibbons ◽  
David T. Yu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Regulatory Elements ◽  
Regulation Of Transcription ◽  
Pol Ii ◽  
Protein Protein Interaction ◽  
Immunological Diseases ◽  
Elongation Step

Transcription by RNA polymerase II (Pol II) is regulated by different processes, including alterations in chromatin structure, interactions between distal regulatory elements and promoters, formation of transcription domains enriched for Pol II and co-regulators, and mechanisms involved in the initiation, elongation, and termination steps of transcription. Transcription factor TFII-I, originally identified as an initiator (INR)-binding protein, contains multiple protein–protein interaction domains and plays diverse roles in the regulation of transcription. Genome-wide analysis revealed that TFII-I associates with expressed as well as repressed genes. Consistently, TFII-I interacts with co-regulators that either positively or negatively regulate the transcription. Furthermore, TFII-I has been shown to regulate transcription pausing by interacting with proteins that promote or inhibit the elongation step of transcription. Changes in TFII-I expression in humans are associated with neurological and immunological diseases as well as cancer. Furthermore, TFII-I is essential for the development of mice and represents a barrier for the induction of pluripotency. Here, we review the known functions of TFII-I related to the regulation of Pol II transcription at the stages of initiation and elongation.

scholarly journals Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

2019 ◽  
Vol 116 (20) ◽  
pp. 9893-9902 ◽  
Author(s):  
Christopher M. Uyehara ◽  
Daniel J. McKay
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Nuclear Receptor ◽  
Binding Sites ◽  
Transcriptional Responses ◽  
Enhancer Activity ◽  
Developmental Transitions ◽  
Experimental Systems ◽  
The Impact

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.

scholarly journals Interplay and cooperation between SREBF1 and master transcription factors regulate lipid metabolism and tumor-promoting pathways in squamous cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Li-Yan Li ◽  
Qian Yang ◽  
Yan-Yi Jiang ◽  
Wei Yang ◽  
Yuan Jiang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Human Cancer ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Gene Set Enrichment Analysis ◽  
Transcriptional Dysregulation

AbstractSquamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC.

Abstract 462: Expression, Phosphorylation and Interaction Partners of the Transcription Factor Grainyhead-like 3 in the Endothelium

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Stefanie Kohlgrüber ◽  
Nadine Dyballa-Rukes ◽  
Sabine Metzger ◽  
Anh Nguyen ◽  
Jim Garmey ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
High Fat Diet ◽  
Ex Vivo ◽  
Src Kinase ◽  
Potential Interaction ◽  
Regulation Of Transcription ◽  
High Fat ◽  
Interaction Partners ◽  
The Impact

The transcription factor Grainyhead-like 3 (GRHL3) regulates apoptosis, migration and NO-bioavailability and thus, critical functions of endothelial cell, which are impaired in many cardiovascular diseases. However, due to the lack of a good antibody, all experiments concerning the regulation of GRHL3 itself were performed on the RNA level. After establishing a new antibody, we analyzed the GRHL3 protein in aortic sections of ApoE-deficient mice fed a high fat diet, a model for atherosclerosis. The diet resulted in a reduction of GRHL3 levels in the endothelium, which was corroborated ex vivo in endothelial cells treated with LDL. This simulated high fat diet also led to a decrease in endothelial NO-synthase. As the activity of transcription factors is regulated by post-translational modifications and protein-protein interactions, we analyzed the phosphorylation of GRHL3 and identified potential interaction partners. Using a combination of immunoprecipitation and -blotting we demonstrated for the first time that GRHL3 is phosphorylated on tyrosine residues. Furthermore, this phosphorylation was NO-inducible and Src-kinase-dependent. After characterization of the modified residues, we will assess their relevance by determining the impact of phospho-mimetic and non-phosphorylatable mutants on functional parameters of endothelial cells. To identify potential interaction partners of GRHL3 we immunoprecipitated the protein and analyzed the co-precipitated proteins by mass spectrometry. We identified the DBHS-proteins NONO and SFPQ, which have been implicated in the regulation of transcription and alternative splicing. The interaction with these two proteins was validated by co-immunoprecipitation. As a next step, we will overexpress and downregulate these proteins in endothelial cells to evaluate their cross-talk with GRHL3. Taken together our findings demonstrate (i) a downregulation of GRHL3 in a disease setting, (ii) a Src-kinase dependent, NO-inducible phosphorylation and (iii) an interaction with other gene-regulatory proteins. The analysis of the functional consequences of these different aspects of GRHL3 regulation will further shed light on the GRHL3 network in the endothelium and thus, its functions in the vasculature.

scholarly journals Multiple positive and negative regulatory elements in the promoter of the mouse homeobox gene Hoxb-4.

1994 ◽  
Vol 14 (12) ◽  
pp. 8143-8154 ◽  
Author(s):  
A Gutman ◽  
J Gilthorpe ◽  
P W Rigby
Keyword(s):  
Mutational Analysis ◽  
Regulatory Element ◽  
Neuroblastoma Cells ◽  
Homeobox Gene ◽  
Regulatory Elements ◽  
Cell Type ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Cell Type Specific ◽  
Specific Regulation

Mouse Hoxb-4 (Hox-2.6) is a homeobox gene that belongs to a family which also includes Hoxa-4, Hoxc-4, and Hoxd-4 and that is related to the Deformed gene in Drosophila melanogaster. We have determined the sequence of 1.2 kb of 5' flanking DNA of mouse Hoxb-4 and by nuclease S1 and primer extension experiments identified two transcription start sites, P1 and P2, 285 and 207 nucleotides upstream of the ATG initiator codon, respectively. We have shown that this region harbors two independent promoters which drive CAT expression in several different cell lines with various efficiencies, suggesting that they are subject to cell-type-specific regulation. Through detailed mutational analysis, we have identified several cis-regulatory elements, located upstream and downstream of the transcription start sites. They include two cell-type-specific negative regulatory elements, which are more active in F9 embryonal carcinoma cells than in neuroblastoma cells (regions a and d at -226 to -186 and +169 to +205, respectively). An additional negative regulatory element has been delimited (region b between +22 and +113). Positive regulation is achieved by binding of HoxTF, a previously unknown factor, to the sequence GCCATTGG (+148 to +155) that is essential for efficient Hoxb-4 expression. We have also defined the minimal promoter sequences and found that they include two 12-bp initiator elements centered around each transcription start site. The complex architecture of the Hoxb-4 promoter provides the framework for fine-tuned transcriptional regulation during embryonic development.

scholarly journals O-GlcNAcylation and O-GlcNAc Cycling Regulate Gene Transcription: Emerging Roles in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1666
Author(s):  
Matthew Parker ◽  
Kenneth Peterson ◽  
Chad Slawson
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Regulatory Elements ◽  
Post Translational Modification ◽  
Hexosamine Biosynthetic Pathway ◽  
Rnap Ii ◽  
Tata Box Binding Protein

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. We will focus on the complex relationship between phosphorylation and O-GlcNAcylation in the regulation of the RNA Polymerase II (RNAP II) pre-initiation complex and the regulation of the carboxyl-terminal domain of RNAP II via the synchronous actions of OGT, OGA, and kinases. Additionally, we discuss how O-GlcNAcylation of TATA-box binding protein (TBP) alters cellular metabolism. Next, in a non-exhaustive manner, we will discuss the current literature on how O-GlcNAcylation drives gene transcription in cancer through changes in transcription factor or chromatin remodeling complex functions. We conclude with a discussion of the challenges associated with studying O-GlcNAcylation and present several new approaches for studying O-GlcNAc regulated transcription that will advance our understanding of the role of O-GlcNAc in cancer.

scholarly journals The elephant shark methylome reveals conservation of epigenetic regulation across jawed vertebrates

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 526 ◽  
Author(s):  
Julian R. Peat ◽  
Oscar Ortega-Recalde ◽  
Olga Kardailsky ◽  
Timothy A. Hore
Keyword(s):  
Epigenetic Modification ◽  
Regulatory Elements ◽  
Cartilaginous Fish ◽  
Critical System ◽  
Modification System ◽  
Elephant Shark ◽  
Transcription Start Sites ◽  
Regulatory Architecture ◽  
Transposon Activity ◽  
And Function

Background: Methylation of CG dinucleotides constitutes a critical system of epigenetic memory in bony vertebrates, where it modulates gene expression and suppresses transposon activity. The genomes of studied vertebrates are pervasively hypermethylated, with the exception of regulatory elements such as transcription start sites (TSSs), where the presence of methylation is associated with gene silencing. This system is not found in the sparsely methylated genomes of invertebrates, and establishing how it arose during early vertebrate evolution is impeded by a paucity of epigenetic data from basal vertebrates. Methods: We perform whole-genome bisulfite sequencing to generate the first genome-wide methylation profiles of a cartilaginous fish, the elephant shark Callorhinchus milii. Employing these to determine the elephant shark methylome structure and its relationship with expression, we compare this with higher vertebrates and an invertebrate chordate using published methylation and transcriptome data.  Results: Like higher vertebrates, the majority of elephant shark CG sites are highly methylated, and methylation is abundant across the genome rather than patterned in the mosaic configuration of invertebrates. This global hypermethylation includes transposable elements and the bodies of genes at all expression levels. Significantly, we document an inverse relationship between TSS methylation and expression in the elephant shark, supporting the presence of the repressive regulatory architecture shared by higher vertebrates. Conclusions: Our demonstration that methylation patterns in a cartilaginous fish are characteristic of higher vertebrates imply the conservation of this epigenetic modification system across jawed vertebrates separated by 465 million years of evolution. In addition, these findings position the elephant shark as a valuable model to explore the evolutionary history and function of vertebrate methylation.

scholarly journals The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

2007 ◽  
Vol 54 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Wiesława Widłak ◽  
Natalia Vydra ◽  
Volha Dudaladava ◽  
Dorota Scieglińska ◽  
Bolesław Winiarski ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Gc Box ◽  
Specific Expression Pattern ◽  
High Level

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

scholarly journals A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

2019 ◽  
Author(s):  
Christopher M. Uyehara ◽  
Daniel J. McKay
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Receptor ◽  
Binding Sites ◽  
Target Genes ◽  
Transcriptional Responses ◽  
Developmental Transitions ◽  
The Impact

ABSTRACTThe ecdysone pathway was amongst the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone-response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.SIGNIFICANCENuclear receptors (NRs) are sequence-specific DNA binding proteins that act as intracellular receptors for small molecules such as hormones. Prior work has shown that NRs function as ligand-dependent switches that initiate a cascade of gene expression changes. The extent to which NRs function as direct regulators of downstream genes in these hierarchies remains incompletely understood. Here, we study the role of the NR EcR in metamorphosis of the Drosophila wing. We find that EcR directly regulates many genes at the top of the hierarchy as well as at downstream genes. Further, we find that EcR binds distinct sets of target genes at different developmental times. This work helps inform how hormones elicit tissue- and temporal-specific responses in target tissues.

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