Transcription imparts architecture, function, and logic to enhancer units

AbstractDistal enhancers remain one of the least understood regulatory elements with pivotal roles in development and disease. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites (TSSs) are robust predictors of enhancer activity with higher resolution and specificity than histone modifications. We show that active enhancer units are precisely delineated by active TSSs, validate that these boundaries are sufficient to capture enhancer function, and confirm that core promoter sequences are required for this activity. Finally, we assay pairs of adjacent units and find that their cumulative activity is best predicted by the strongest unit within the pair. Synthetic fusions of enhancer units demonstrate that adjacency imposes winner-takes-all logic, revealing a simple design for a maximum-activity filter of enhancer unit outputs. Together, our results define fundamental enhancer units and a principle of non-cooperativity between adjacent units.

Download Full-text

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming

10.21203/rs.3.rs-384560/v1 ◽

2021 ◽

Author(s):

Alexandre Gaspar-Maia ◽

Wazim Mohammed Ismail ◽

Amelia Mazzone ◽

Jagneet Kaur ◽

Stephanie Safgren ◽

...

Keyword(s):

Regulatory Elements ◽

Histone Variants ◽

Cellular Reprogramming ◽

Negative Regulator ◽

Cellular Heterogeneity ◽

Enhancer Activity ◽

Active Enhancer ◽

A Cell ◽

Cell Type Specific

Abstract Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macroH2A-Bound Enhancers’, that negatively modulate enhancer activity. We find macroH2A variants enriched at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and its repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling, we show that the loss of macroH2A2 leads to increased cellular heterogeneity that may help to explain the role of macroH2A variants in defining oncogenic transcriptional dependencies.

Download Full-text

Systematic dissection of genomic features determining transcription factor binding and enhancer function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621150114 ◽

2017 ◽

Vol 114 (7) ◽

pp. E1291-E1300 ◽

Cited By ~ 74

Author(s):

Sharon R. Grossman ◽

Xiaolan Zhang ◽

Li Wang ◽

Jesse Engreitz ◽

Alexandre Melnikov ◽

...

Keyword(s):

Regulatory Elements ◽

Chromatin Accessibility ◽

Binding Assays ◽

Genomic Locus ◽

Enhancer Activity ◽

Genomic Features ◽

Motif Site ◽

Reporter Assays ◽

Enhancer Function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

Download Full-text

Resolving systematic errors in widely-used enhancer activity assays in human cells enables genome-wide functional enhancer characterization

10.1101/164590 ◽

2017 ◽

Cited By ~ 4

Author(s):

Felix Muerdter ◽

Łukasz M. Boryń ◽

Ashley R. Woodfin ◽

Christoph Neumayr ◽

Martina Rath ◽

...

Keyword(s):

Mammalian Cells ◽

Core Promoter ◽

Human Cells ◽

Type I ◽

Enhancer Activity ◽

Genome Wide ◽

Bacterial Plasmid ◽

Reporter Assays ◽

Hela S3 ◽

Plasmid Transfection

AbstractThe identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliable: (1) the function of the bacterial plasmid origin-of-replication (ORI) as a conflicting core-promoter and (2) the activation of a type I interferon (IFN-I) response. These problems cause strongly confounding false-positives and -negatives in luciferase assays and genome-wide STARR-seq screens. We overcome both problems by directly employing the ORI as a core-promoter and by inhibiting two kinases central to IFN-I induction. This corrects luciferase assays and enables genome-wide STARR-seq screens in human cells. Comprehensive enhancer activity profiles in HeLa-S3 cells uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells, and are key to the characterization of human enhancers.

Download Full-text

Recent advances in high-throughput approaches to dissect enhancer function

F1000Research ◽

10.12688/f1000research.11581.1 ◽

2017 ◽

Vol 6 ◽

pp. 939 ◽

Cited By ~ 15

Author(s):

David Santiago-Algarra ◽

Lan T.M. Dao ◽

Lydie Pradel ◽

Alexandre España ◽

Salvatore Spicuglia

Keyword(s):

High Throughput ◽

Regulatory Elements ◽

Genetic Alterations ◽

Regulatory Sequences ◽

Transcription Start ◽

Enhancer Activity ◽

Recent Advances ◽

Reporter Assays ◽

Enhancer Function ◽

Higher Eukaryotes

The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.

Download Full-text

A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

10.1101/061606 ◽

2016 ◽

Cited By ~ 1

Author(s):

Fumitaka Inoue ◽

Martin Kircher ◽

Beth Martin ◽

Gregory M. Cooper ◽

Daniela M. Witten ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Sequence Information ◽

Regulatory Sequences ◽

Enhancer Activity ◽

Functional Activities ◽

Reporter Assays ◽

Bona Fide ◽

Massively Parallel Reporter Assay

AbstractCandidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can drive expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences incis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this question in a systematic manner, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2,236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally-based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson’s R2of 0.347 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for howcis-regulatory elements are identified, prioritized and functionally validated.

Download Full-text

Determinants of Transcription Initiation Directionality in Metazoans

10.1101/224642 ◽

2017 ◽

Author(s):

Mahmoud M. Ibrahim ◽

Aslihan Karabacak ◽

Alexander Glahs ◽

Ena Kolundzic ◽

Antje Hirsekorn ◽

...

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Regulatory Element ◽

Promoter Sequence ◽

Regulatory Elements ◽

Integrative Model ◽

Promoter Regions ◽

Promoter Sequences ◽

C Elegans ◽

Divergent Transcription

AbstractDivergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general and passive feature of all eukaryotic cis regulatory elements. To address this, we define promoters and enhancers in C. elegans, D. melanogaster and H. sapiens using ATAC-Seq and investigate the determinants of their transcription initiation directionalities by analyzing genome-wide nascent, cap-selected, polymerase run-on assays. All three species initiate divergent transcription from separate core promoter sequences. Sequence asymmetry downstream of forward and reverse initiation sites, known to be important for termination and stability in H. sapiens, is unique in each species. Chromatin states of divergent promoters are not entirely conserved, but in all three species, the levels of histone modifications on the +1 nucleosome are independent from those on the -1 nucleosome, arguing for independent initiation events. This is supported by an integrative model of H3K4me3 levels and core promoter sequence that is highly predictive of promoter directionality and of two types of promoters: those with balanced initiation directionality and those with skewed directionality. Lastly, D. melanogaster enhancers display variation in chromatin architecture depending on enhancer location, and D. melanogaster promoter regions with dual enhancer/promoter potential are enriched for divergent transcription. Our results point to a high degree of variation in regulatory element transcription initiation directionality within and between metazoans, and to non-passive regulatory mechanisms of transcription initiation directionality in those species.

Download Full-text

RAEdb: a database of enhancers identified by high-throughput reporter assays

Database ◽

10.1093/database/bay140 ◽

2019 ◽

Vol 2019 ◽

Cited By ~ 4

Author(s):

Zena Cai ◽

Ya Cui ◽

Zhiying Tan ◽

Gaihua Zhang ◽

Zhongyang Tan ◽

...

Keyword(s):

Cell Line ◽

High Throughput ◽

Human Cell ◽

Regulatory Elements ◽

The Other ◽

Activity Score ◽

Human Cell Lines ◽

Enhancer Activity ◽

A Genome ◽

Reporter Assays

Abstract High-throughput reporter assays have been recently developed to directly and quantitatively assess enhancer activity for thousands of regulatory elements. However, there is still no database to collect these enhancers. We developed RAEdb, the first database to collect enhancers identified by high-throughput reporter assays. RAEdb includes 538 320 enhancers derived from eight studies, most of which were from six human cell lines. An activity score was assigned to each enhancer based on reporter assays. Based on these enhancers, 7658 epromoters (promoters with enhancer activity) were identified and stored in the database. RAEdb provides two ways of searches: the first is to search studies by species and cell line; the other is to search enhancers or epromoters by position, activity score, sequence and gene. RAEdb also provides a genome browser to query, visualize and compare enhancers. All data in RAEdb is freely available for download.

Download Full-text

Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences

International Journal of Molecular Sciences ◽

10.3390/ijms22115704 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5704

Author(s):

Xiaofan Feng ◽

Mario Andrea Marchisio

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Tata Box ◽

Yeast Saccharomyces Cerevisiae ◽

Transcription Start Sites ◽

Core Promoters ◽

Promoter Sequences ◽

Synthetic Promoters ◽

Synthetic Gene Circuits ◽

Enhance Protein Synthesis

Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.

Download Full-text

Regulatory Elements of the Staphylococcus aureus Protein A (Spa) Promoter

Journal of Bacteriology ◽

10.1128/jb.186.12.3738-3748.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3738-3748 ◽

Cited By ~ 50

Author(s):

Jinxin Gao ◽

George C. Stewart

Keyword(s):

Staphylococcus Aureus ◽

Growth Phase ◽

Core Promoter ◽

Consensus Sequence ◽

Protein A ◽

Promoter Sequence ◽

Regulatory Elements ◽

Exponential Growth Phase ◽

Staphylococcal Protein A ◽

Promoter Sequences

ABSTRACT Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3′ end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription.

Download Full-text

Intron-driven gene expression in the absence of a core promoter in Arabidopsis thaliana

10.1101/083261 ◽

2016 ◽

Author(s):

Jenna E Gallegos ◽

Alan B Rose

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Core Promoter ◽

Regulatory Elements ◽

Intron Position ◽

Mrna Accumulation ◽

Practical Applications ◽

Promoter Sequences ◽

The Core

AbstractIn diverse eukaryotes, certain introns increase mRNA accumulation through the poorly understood mechanism of intron-mediated enhancement (IME). A distinguishing feature of IME is that these introns have no effect from upstream or more than 1 Kb downstream of the transcription start site (TSS). To more precisely define the intron position requirements for IME in Arabidopsis, we tested the effect of the UBQ10 intron on gene expression from 6 different positions surrounding the TSS of a TRP1:GUS fusion. The intron strongly increased expression from all transcribed positions, but had no effect when 204 nt or more upstream of the 5’-most TSS. When the intron was located in the 5’ UTR, the TSS unexpectedly changed, resulting in longer transcripts. Remarkably, deleting 303 nt of the core promoter, including all known TSS’s and all but 18 nt of the 5’ UTR, had virtually no effect on the level of gene expression as long as a stimulating intron was included in the gene. When the core promoter was deleted, transcription initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. Together, these results suggest that certain introns play unexpectedly large roles in directing transcription initiation and represent a previously unrecognized type of downstream regulatory elements for genes transcribed by RNA polymerase II. This study also demonstrates considerable flexibility in the sequences surrounding the TSS, indicating that the TSS is not determined by promoter sequences alone. These findings are relevant in practical applications where introns are used to increase gene expression and contribute to our general understanding of gene structure and regulation in eukaryotes.

Download Full-text