scholarly journals CTCF blocks anti-sense transcription initiation at divergent gene promoters

2021 ◽  
Author(s):  
Jing Luan ◽  
Camille M. Syrett ◽  
Marit W. Vermunt ◽  
Allison Cote ◽  
Jacob M. Tome ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Great Majority ◽  
Antisense Transcription ◽  
Ctcf Binding ◽  
Gene Promoters ◽  
Core Promoters ◽  
Rna Fish ◽  
Divergent Gene ◽  
Regulated Competition ◽  
Transcriptional Landscape

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

scholarly journals Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities

2017 ◽  
Author(s):  
Sarah Rennie ◽  
Maria Dalby ◽  
Marta Lloret-Llinares ◽  
Stylianos Bakoulis ◽  
Christian Dalager Vaagensø ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Core Promoter ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Chromatin Interaction ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Core Promoters ◽  
Promoter Architecture ◽  
Regulatory Functions

ABSTRACTMammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

scholarly journals CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Françoise S Howe ◽  
Andrew Russell ◽  
Anna R Lamstaes ◽  
Afaf El-Sagheer ◽  
Anitha Nair ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Antisense Transcript ◽  
Transcription Termination ◽  
Antisense Transcription ◽  
Eukaryotic Cells ◽  
Dna Templates ◽  
Template Strand ◽  
Guide Rnas ◽  
Genetic Interventions ◽  
Transcriptional Landscape

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

scholarly journals Antisense lncRNA transcription drives stochastic Protocadherin α promoter choice

2018 ◽  
Author(s):  
Daniele Canzio ◽  
Chiamaka L. Nwakeze ◽  
Adan Horta ◽  
Sandy M. Rajkumar ◽  
Eliot L. Coffey ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Neural Circuit ◽  
Antisense Transcription ◽  
Dna Demethylation ◽  
Ctcf Binding ◽  
Gene Promoters ◽  
Protein Coding ◽  
Close Proximity ◽  
A Cell

SUMMARYStochastic and combinatorial activation of clustered Protocadherin (Pcdh) α, β, and γ gene promoters generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here we show that Pcdhα promoter choice requires transcription of a long noncoding RNA (lncRNA) initiated from newly identified promoters located in the protein coding sequence of each Pcdhα exon. Antisense transcription of the lncRNA through the sense promoter results in its activation and in DNA demethylation of the binding sites for the CCCTC-binding protein, CTCF, located in close proximity to both sense and antisense promoters. Increased CTCF binding promotes the assembly of long-range DNA contacts between the activated promoter and a neuron-specific enhancer, thus locking in the epigenetic state of the stochastically chosen Pcdhα promoter. Examination of this hierarchical molecular mechanism in differentiating olfactory sensory neurons, suggests that antisense Pcdhα transcription is a key prerequisite for stochastic Pcdhα promoter choice in vivo.

scholarly journals CRISPRi is not strand-specific and redefines the transcriptional landscape

2017 ◽  
Author(s):  
Françoise S. Howe ◽  
Andrew Russell ◽  
Afaf El-Sagheer ◽  
Anitha Nair ◽  
Tom Brown ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Antisense Transcript ◽  
Transcription Termination ◽  
Antisense Transcription ◽  
Eukaryotic Cells ◽  
Dna Templates ◽  
Template Strand ◽  
Guide Rnas ◽  
Genetic Interventions ◽  
Transcriptional Landscape

ABSTRACTCRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, binding of sgRNA/dCas9 creates an environment that is permissive for transcription initiation and termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9 binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

scholarly journals Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I

2015 ◽  
Vol 112 (7) ◽  
pp. E677-E686 ◽  
Author(s):  
Rodrigo Peña-Hernández ◽  
Maud Marques ◽  
Khalid Hilmi ◽  
Teijun Zhao ◽  
Amine Saad ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Cellular Response ◽  
Target Genes ◽  
Regulatory Protein ◽  
Metabolic Stress ◽  
Ctcf Binding ◽  
Gene Promoters ◽  
The Impact

CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.

scholarly journals Antisense-mediated repression of SAGA-dependent genes involves the HIR histone chaperone

2021 ◽  
Author(s):  
Julien Soudet ◽  
Nissrine Beyrouthy ◽  
Anna Marta Pastucha ◽  
Andrea Maffioletti ◽  
Zahra Bakir ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
De Novo ◽  
Antisense Transcription ◽  
Histone Chaperone ◽  
Gene Promoters ◽  
Genome Wide ◽  
Chaperone Complex ◽  
Histone Deposition ◽  
Transcription Interference

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae (S. cerevisiae), TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.

The Long Road to Understanding RNAPII Transcription Initiation and Related Syndromes

2021 ◽  
Vol 90 (1) ◽  
pp. 193-219
Author(s):  
Emmanuel Compe ◽  
Jean-Marc Egly
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Regulatory Elements ◽  
Initiation Mechanism ◽  
Protein Coding ◽  
Core Promoters ◽  
General Transcription Factors

In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.

Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region

1986 ◽  
Vol 6 (12) ◽  
pp. 4458-4466
Author(s):  
D E Ingolia ◽  
M R Al-Ubaidi ◽  
C Y Yeung ◽  
H A Bigo ◽  
D Wright ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Gene Promoter ◽  
Mouse Cell ◽  
Gene Promoters ◽  
Sp1 Transcription Factor ◽  
Base Pair Fragment ◽  
Functional Analyses

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

scholarly journals p53 dynamically directs TFIID assembly on target gene promoters

2016 ◽  
Author(s):  
R. A. Coleman ◽  
Z. Qiao ◽  
S. K. Singh ◽  
C. S. Peng ◽  
M. Cianfrocco ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Gene Networks ◽  
Target Gene ◽  
Core Promoter ◽  
Gene Promoters ◽  
Binding Modes ◽  
Dissociation Kinetics ◽  
Target Promoters

AbstractThe p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

scholarly journals The Transcriptional landscape of Streptococcus pneumoniae reveals a complex operon architecture and abundant riboregulation critical for growth and virulence

2018 ◽  
Author(s):  
Indu Warrier ◽  
Nikhil Ram-Mohan ◽  
Zeyu Zhu ◽  
Ariana Hazery ◽  
Michelle M Meyer ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Throughput ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Regulatory Element ◽  
Fine Tuning ◽  
Transcriptional Units ◽  
Rna Elements ◽  
Transcriptional Landscape

AbstractEfficient and highly organized transcription initiation and termination is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Over the last decade, our simplistic outlook of bacterial transcriptional regulation and architecture has evolved to include stimulus-responsive regulation by untranslated RNA and the formation of alternate transcriptional units. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae by applying a combination of high-throughput RNA-sequencing techniques. Our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal transcription start sites (TSS) and transcription terminators (TTS) suggesting that more fine-tuning of regulation occurs than previously thought. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR) of genes. By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to determine the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-) regulators to mitigate virulence and antibiotic resistance.

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