Faculty Opinions recommendation of Genome-wide quantitative enhancer activity maps identified by STARR-seq.

AbstractAdolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is the most common pediatric musculoskeletal disorder, affecting ∼3% of the population worldwide. However, its genetic bases and tissues of origin remain largely unknown. Several genome-wide association studies (GWAS) have implicated nucleotide variants in noncoding sequences that control genes with important roles in cartilage, muscle, bone, connective tissue and intervertebral discs (IVDs) as drivers of AIS susceptibility. Here, we set out to define the expression of AIS-associated genes and active regulatory elements by performing RNA-seq and ChIP-seq against H3K27ac in these tissues in mouse and human. Our study highlights genetic pathways involving AIS-associated loci that regulate chondrogenesis, IVD development and connective tissue maintenance and homeostasis. In addition, we identify thousands of putative AIS-associated regulatory elements which may orchestrate tissue-specific expression in musculoskeletal tissues of the spine. Quantification of enhancer activity of several candidate regulatory elements from our study identifies three functional enhancers carrying AIS-associated GWAS SNPs at the ADGRG6 and BNC2 loci. Our findings provide a novel genome-wide catalog of AIS-relevant genes and regulatory elements and aid in the identification of novel targets for AIS causality and treatment.

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Genome-wide discovery of SLE genetic risk variant allelic enhancer activity

10.1101/2020.01.20.906701 ◽

2020 ◽

Cited By ~ 3

Author(s):

Xiaoming Lu ◽

Xiaoting Chen ◽

Carmy Forney ◽

Omer Donmez ◽

Daniel Miller ◽

...

Keyword(s):

Lupus Erythematosus ◽

Association Studies ◽

Binding Motif ◽

Genome Wide Association Studies ◽

Genomic Context ◽

Transcriptional Enhancer ◽

Enhancer Activity ◽

Risk Variant ◽

Genome Wide ◽

Depth Analysis

AbstractGenome-wide association studies of Systemic Lupus Erythematosus (SLE) nominate 3,073 genetic variants at 91 risk loci. To systematically screen these variants for allelic transcriptional enhancer activity, we constructed a massively parallel reporter assay (MPRA) library comprising 12,396 DNA oligonucleotides containing the genomic context around every allele of each SLE variant. Transfection into EBV-infected B cells revealed 482 variants with enhancer activity, with 51 variants showing genotype-dependent (allelic) enhancer activity at 27 risk loci. In-depth analysis of allelic transcription factor (TF) binding at and around these 51 variants identified one class of TFs whose DNA-binding motif tends to be directly altered by the risk variant and a second, larger class of TFs that also bind allelically but do not have their motifs directly altered by the variant. Collectively, our approach provides a blueprint for the discovery of allelic gene regulation at risk loci for any disease and offers insight into the transcriptional regulatory mechanisms underlying SLE.

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Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo

eLife ◽

10.7554/elife.08445 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 21

Author(s):

Rupinder Sayal ◽

Jacqueline M Dresch ◽

Irina Pushel ◽

Benjamin R Taylor ◽

David N Arnosti

Keyword(s):

Drosophila Embryo ◽

Enhancer Activity ◽

Genome Wide ◽

Regulatory Machinery ◽

A Genome ◽

Early Drosophila Embryo ◽

Wide Scale ◽

Factor Interactions ◽

Identification And Characterization ◽

Enhancer Identification

Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale.

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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.

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Characterizing the function of EPB41L4A in the predisposition to papillary thyroid carcinoma

Scientific Reports ◽

10.1038/s41598-020-76606-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Daniel F. Comiskey ◽

Huiling He ◽

Sandya Liyanarachchi ◽

Mehek S. Sheikh ◽

Isabella V. Hendrickson ◽

...

Keyword(s):

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Genome Wide Association Study ◽

High Linkage Disequilibrium ◽

Papillary Thyroid ◽

Enhancer Activity ◽

Enhancer Region ◽

Protective Allele ◽

Genome Wide ◽

Underlying Causes

AbstractPapillary thyroid carcinoma (PTC) is the most common histotype of thyroid carcinoma. The heritability of PTC is high compared to other cancers, but its underlying causes are unknown. A recent genome-wide association study revealed the association of a variant at the 5q22 locus, rs73227498, with PTC predisposition. We report that rs17134155, a variant in high linkage disequilibrium with rs73227498, is located in an enhancer region downstream of coding transcripts of EPB41L4A. Rs17134155 showed significant enhancer activity in luciferase assays, and haplotypes containing the protective allele of this variant conferred a significantly lower risk of PTC. While the index SNP, rs73227498, acted as a significant cis-eQTL for expression of EPB41L4A, rs17134155 was a significant cis-sQTL for the alternative splicing of a non-coding transcript of EPB41L4A, called EPB41L4A-203. We also performed knockdown of EPB41L4A followed by microarray analysis. Some of the top differentially-expressed genes were represented among regulators of the WNT/β-catenin signaling pathway. Our results indicate that an enhancer region at 5q22 regulates the expression and splicing of EPB41L4A transcripts. We also provide evidence that EPB41L4A expression is involved in regulating growth and differentiation pathways, suggesting that decreased expression of EPB41L4A is a mechanism in the predisposition to PTC.

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Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale

Genome ◽

10.1139/gen-2020-0104 ◽

2020 ◽

pp. 1-23

Author(s):

Ian C. Tobias ◽

Luis E. Abatti ◽

Sakthi D. Moorthy ◽

Shanelle Mullany ◽

Tiegh Taylor ◽

...

Keyword(s):

High Throughput Screening ◽

Target Genes ◽

Regulatory Sequences ◽

Enhancer Activity ◽

Reading Frame ◽

Enhancer Prediction ◽

Genome Wide ◽

A Genome ◽

Enhancer Function

Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries, and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements, and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states, and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

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Identification and characterization of Hoxa9 binding sites in hematopoietic cells

Blood ◽

10.1182/blood-2011-03-341081 ◽

2012 ◽

Vol 119 (2) ◽

pp. 388-398 ◽

Cited By ~ 119

Author(s):

Yongsheng Huang ◽

Kajal Sitwala ◽

Joel Bronstein ◽

Daniel Sanders ◽

Monisha Dandekar ◽

...

Keyword(s):

Transcriptional Activation ◽

Binding Sites ◽

Enhancer Activity ◽

Transcription Start Sites ◽

Genome Wide ◽

A Genome ◽

Evolutionarily Conserved ◽

P300 Binding ◽

Histone H3k4

The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

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TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity

Journal of Virology ◽

10.1128/jvi.00513-19 ◽

2019 ◽

Vol 93 (16) ◽

Cited By ~ 2

Author(s):

Chong Wang ◽

Sizun Jiang ◽

Luyao Zhang ◽

Difei Li ◽

Jun Liang ◽

...

Keyword(s):

Epstein Barr Virus ◽

Target Genes ◽

Fluorescent Protein ◽

Simian Virus 40 ◽

Enhancer Activity ◽

Barr Virus ◽

Reporter Activity ◽

Genome Wide ◽

A Genome ◽

Epstein Barr

ABSTRACTSuper-enhancers (SEs) are clusters of enhancers marked by extraordinarily high and broad chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-κB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESE function, MYC ESEs were cloned upstream of a green fluorescent protein (GFP) reporter. Reporters driven by MYC ESEs 525 kb and 428 kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE-driven luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TBP-associated factor (TAF) family proteins, including TAF8, TAF11, and TAF3, were essential for the activity of the integrated 525ESE-driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout and restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes, including the ETS1 and BCL2 genes. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.IMPORTANCESEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not simian virus 40 (SV40) enhancer. These proteins not only were essential for the reporter activity but also were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.

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A functional genomics pipeline identifies pleiotropy and cross-tissue effects within obesity-associated GWAS loci

Nature Communications ◽

10.1038/s41467-021-25614-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Amelia C. Joslin ◽

Débora R. Sobreira ◽

Grace T. Hansen ◽

Noboru J. Sakabe ◽

Ivy Aneas ◽

...

Keyword(s):

Association Studies ◽

Complex Model ◽

Genome Wide Association Studies ◽

Enhancer Activity ◽

Chromatin Interactions ◽

Genome Wide ◽

Causal Variants ◽

Cell Type Specific ◽

Gene Regulatory ◽

Massively Parallel Reporter Assay

AbstractGenome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS.

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Bromodomain protein BRDT directs ΔNp63 function and super-enhancer activity in a subset of esophageal squamous cell carcinomas

Cell Death and Differentiation ◽

10.1038/s41418-021-00751-w ◽

2021 ◽

Author(s):

Xin Wang ◽

Ana P. Kutschat ◽

Moyuru Yamada ◽

Evangelos Prokakis ◽

Patricia Böttcher ◽

...

Keyword(s):

Squamous Cell ◽

Target Genes ◽

Experimental Studies ◽

Specific Protein ◽

Chromatin Interaction ◽

Cell Phenotype ◽

Precision Oncology ◽

Enhancer Activity ◽

Genome Wide ◽

Geographical Regions

AbstractEsophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer with a particularly high prevalence in certain geographical regions and a poor prognosis with a 5-year survival rate of 15–25%. Despite numerous studies characterizing the genetic and transcriptomic landscape of ESCC, there are currently no effective targeted therapies. In this study, we used an unbiased screening approach to uncover novel molecular precision oncology targets for ESCC and identified the bromodomain and extraterminal (BET) family member bromodomain testis-specific protein (BRDT) to be uniquely expressed in a subgroup of ESCC. Experimental studies revealed that BRDT expression promotes migration but is dispensable for cell proliferation. Further mechanistic insight was gained through transcriptome analyses, which revealed that BRDT controls the expression of a subset of ΔNp63 target genes. Epigenome and genome-wide occupancy studies, combined with genome-wide chromatin interaction studies, revealed that BRDT colocalizes and interacts with ΔNp63 to drive a unique transcriptional program and modulate cell phenotype. Our data demonstrate that these genomic regions are enriched for super-enhancers that loop to critical ΔNp63 target genes related to the squamous phenotype such as KRT14, FAT2, and PTHLH. Interestingly, BET proteolysis-targeting chimera, MZ1, reversed the activation of these genes. Importantly, we observed a preferential degradation of BRDT by MZ1 compared with BRD2, BRD3, and BRD4. Taken together, these findings reveal a previously unknown function of BRDT in ESCC and provide a proof-of-concept that BRDT may represent a novel therapeutic target in cancer.

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