RSC-mediated nucleosome positioning and GRFs form barriers in promoters to limit divergent noncoding transcription

The directionality of gene promoters - the ratio of protein-coding over divergent noncoding transcription - is highly variable and regulated. How promoter directionality is controlled remains poorly understood. We show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription. Depletion of RSC increased divergent noncoding transcription and decreased protein-coding transcription at promoters with strong directionality. Consistent with RSCs role in regulating chromatin, RSC depletion impacts nucleosome occupancy upstream of the nucleosome depleted region where divergent transcription initiates, suggesting that nucleosome positioning at the 5 prime border of gene promoters physically blocks the recruitment of the transcription machinery and inhibits initiation of divergent transcription. Highly directional promoters were also enriched for the binding of GRFs such as Reb1 and Abf1. Furthermore, ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 protein can suppress divergent transcription. Our data suggest that RSC-mediated nucleosome positioning and GRFs play a pervasive role in repressing divergent transcription. We propose that any DNA binding factor, when stably associated with cryptic transcription start sites, can form a barrier for repressing divergent transcription. Our study provides an explanation as to why certain promoters are more directional than others.

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Repression of divergent noncoding transcription by a sequence-specific transcription factor

10.1101/314310 ◽

2018 ◽

Author(s):

Andrew CK Wu ◽

Harshil Patel ◽

Minghao Chia ◽

Fabien Moretto ◽

David Frith ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Initiation ◽

Large Fraction ◽

Regulatory Elements ◽

Gene Promoters ◽

Specific Transcription Factor ◽

Highly Expressed Genes ◽

Divergent Transcription ◽

Noncoding Transcription ◽

Cryptic Promoters

SummaryMany active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1 regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeller. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality towards productive transcription.

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High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.617998 ◽

2021 ◽

Vol 10 ◽

Author(s):

Benedikt M. Markus ◽

Benjamin S. Waldman ◽

Hernan A. Lorenzi ◽

Sebastian Lourido

Keyword(s):

Toxoplasma Gondii ◽

Transcription Initiation ◽

Nucleosome Occupancy ◽

Mrna Isoforms ◽

Protein Coding ◽

Transcription Start Sites ◽

Promoter Architecture ◽

Depth Analysis ◽

Life Threatening ◽

Specific Alternative

Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

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Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.2937 ◽

1991 ◽

Vol 11 (6) ◽

pp. 2937-2945 ◽

Cited By ~ 43

Author(s):

E Martinez ◽

Y Dusserre ◽

W Wahli ◽

N Mermod

Keyword(s):

Estrogen Receptor ◽

Dna Binding ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Limiting Factor ◽

Gene Promoters ◽

Protein Coding ◽

Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

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Transcription initiation mapping in 31 bovine tissues reveals complex promoter activity, pervasive transcription, and tissue-specific promoter usage

10.1101/2020.09.05.284547 ◽

2020 ◽

Author(s):

D.E. Goszczynski ◽

M.M. Halstead ◽

A.D. Islas-Trejo ◽

H. Zhou ◽

P.J. Ross

Keyword(s):

Transcription Initiation ◽

Promoter Activity ◽

Bovine Genome ◽

Transcription Start ◽

Protein Coding ◽

Tissue Specific ◽

Transcription Start Sites ◽

Expression Control ◽

Tissue Specific Promoter ◽

Genome Annotations

ABSTRACTCharacterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and includes a low number of transcripts in comparison to human and mouse. Using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE data for producing a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-Seq. Lastly, gene co-expression networks revealed an impressive use of tissue-specific promoters, especially between brain and testicle, which expressed several genes in common from alternate transcription start sites. Regions surrounding co-expressed modules were enriched in binding factor motifs representative of their tissues. This annotation will be highly useful for future studies on expression control in cattle and other species. Furthermore, these data provide significant insight into transcriptional activity for a comprehensive set of tissues.

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The origin and evolution of a distinct mechanism of transcription initiation in yeasts

10.1101/2020.04.04.025502 ◽

2020 ◽

Cited By ~ 3

Author(s):

Zhaolian Lu ◽

Zhenguo Lin

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

Core Promoter ◽

Model Species ◽

Initiation Mechanism ◽

Protein Coding ◽

Origin And Evolution ◽

Transcription Start Sites ◽

Mrna Maturation ◽

Nucleotide Resolution

ABSTRACTThe molecular process of transcription by RNA Polymerase II is highly conserved among eukaryotes (“classic model”). Intriguingly, a distinct way of locating transcription start sites (TSSs) was found in a budding yeast Saccharomyces cerevisiae (“scanning model”). The origin of the “scanning model” and its underlying genetic mechanisms remain unsolved. Herein, we applied genomic approaches to address these questions. We first identified TSSs at a single-nucleotide resolution for 12 yeast species using the nAnT-iCAGE technique, which significantly improved the annotations of these genomes by providing accurate 5’boundaries of protein-coding genes. We then infer the initiation mechanism of a species based on its TSS maps and genome sequences. We found that the “scanning model” had originated after the split of Yarrowia lipolytica and the rest of budding yeasts. An adenine-rich region immediately upstream of TSS had appeared during the evolution of the “scanning model” species, which might facilitate TSS selection in these species. Both initiation mechanisms share a strong preference for pyrimidine-purine dinucleotides surrounding the TSS. Our results suggested that the purine is required for accurately recruiting the first nucleotide, increasing the chance of being capped during mRNA maturation, which is critical for efficient translation initiation. Based on our findings, we proposed a model of TSS selection for the “scanning model” species. Besides, our study also demonstrated that the intrinsic sequence feature primarily determines the distribution of initiation activities within a core promoter (core promoter shape).

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The NSL complex mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise

10.1101/419408 ◽

2018 ◽

Author(s):

Kin Chung Lam ◽

Ho-Ryun Chung ◽

Giuseppe Semplicio ◽

Vivek Bhardwaj ◽

Shantanu S. Iyer ◽

...

Keyword(s):

Transcription Initiation ◽

Dependent Manner ◽

Gene Promoters ◽

Transcription Start ◽

Transcription Start Sites ◽

Chromatin Remodeling Complex ◽

Selection For ◽

Necessary And Sufficient ◽

Target Promoters ◽

Active Genes

AbstractNucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDR and the precise positioning of the +1 nucleosome is maintained on active genes remains unclear. Here, we report that the Drosophila Non-Specific Lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. The NSL complex is not only essential for transcription but is required for accurate TSS selection for genes with multiple TSSs. Further, loss of NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

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Divergent Transcription from Active Promoters

Science ◽

10.1126/science.1162253 ◽

2008 ◽

Vol 322 (5909) ◽

pp. 1849-1851 ◽

Cited By ~ 573

Author(s):

Amy C. Seila ◽

J. Mauro Calabrese ◽

Stuart S. Levine ◽

Gene W. Yeo ◽

Peter B. Rahl ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

Northern Analysis ◽

Gene Promoters ◽

Transcription Start ◽

Promoter Regions ◽

Short Rnas ◽

Protein Encoding ◽

Divergent Transcription ◽

Encoding Gene

Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site–associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.

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Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

10.1101/055731 ◽

2016 ◽

Author(s):

Yun Chen ◽

Athma A. Pai ◽

Jan Herudek ◽

Michal Lubas ◽

Nicola Meola ◽

...

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Building Blocks ◽

Gene Promoters ◽

Mrna Transcription ◽

Transcription Start ◽

Transcription Start Sites ◽

Alternative Transcription ◽

Mammalian Genomes ◽

Polyadenylation Sites

AbstractMammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes.

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Nucleosome Organization around Pseudogenes in the Human Genome

BioMed Research International ◽

10.1155/2015/821596 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Guoqing Liu ◽

Fen Feng ◽

Xiujuan Zhao ◽

Lu Cai

Keyword(s):

Gene Expression ◽

Experimental Data ◽

Human Genome ◽

Genome Evolution ◽

Transcription Initiation ◽

Transcriptional Activity ◽

Nucleosome Occupancy ◽

Nucleosome Positioning ◽

Consistent Pattern ◽

Nucleosome Organization

Pseudogene, disabled copy of functional gene, plays a subtle role in gene expression and genome evolution. The first step in deciphering RNA-level regulation of pseudogenes is to understand their transcriptional activity. So far, there has been no report on possible roles of nucleosome organization in pseudogene transcription. In this paper, we investigated the effect of nucleosome positioning on pseudogene transcription. For transcribed pseudogenes, the experimental nucleosome occupancy shows a prominent depletion at the regions both upstream of pseudogene start positions and downstream of pseudogene end positions. Intriguingly, the same depletion is also observed for nontranscribed pseudogenes, which is unexpected since nucleosome depletion in those regions is thought to be unnecessary in light of the nontranscriptional property of those pseudogenes. The sequence-dependent prediction of nucleosome occupancy shows a consistent pattern with the experimental data-based analysis. Our results indicate that nucleosome positioning may play important roles in both the transcription initiation and termination of pseudogenes.

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