Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10−15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C).Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk “C” allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889.Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

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Disruption of c-MYC binding and chromosomal looping involving genetic variants associated with ankylosing spondylitis upstream of RUNX3 promoter

10.1101/2021.09.07.459234 ◽

2021 ◽

Author(s):

Carla J Cohen ◽

Connor Davidson ◽

Carlo Selmi ◽

Paul Bowness ◽

Julian C Knight ◽

...

Keyword(s):

Ankylosing Spondylitis ◽

Binding Site ◽

Regulatory Elements ◽

Published Data ◽

Open Chromatin ◽

Physical Interaction ◽

Nucleotide Polymorphisms ◽

Genetic Associations ◽

Chromosome Conformation ◽

Nuclear Extracts

ABSTRACTBackgroundAnkylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p= 2.6 x 10-15) lies ~13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C).ResultsInterrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes in contrast to Jurkat T cell line or primary T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 there was consistently less binding to the AS-risk “C” allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n=2; p<0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889.ConclusionsThe lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

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Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2653 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2653-2659 ◽

Cited By ~ 33

Author(s):

D Kardassis ◽

M Hadzopoulou-Cladaras ◽

D P Ramji ◽

R Cortese ◽

V I Zannis ◽

...

Keyword(s):

Binding Site ◽

Regulatory Elements ◽

Apob Gene ◽

Mobility Shift ◽

Promoter Elements ◽

Nuclear Extracts ◽

Dna Binding Site ◽

Gene Definition ◽

Definition Of ◽

Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

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Association study of genes related to bone formation and resorption and the extent of radiographic change in ankylosing spondylitis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2013-204835 ◽

2014 ◽

Vol 74 (7) ◽

pp. 1387-1393 ◽

Cited By ~ 41

Author(s):

A Cortes ◽

W P Maksymowych ◽

B P Wordsworth ◽

R D Inman ◽

P Danoy ◽

...

Keyword(s):

New York ◽

Ankylosing Spondylitis ◽

Phase 1 ◽

Nucleotide Polymorphisms ◽

Genetic Associations ◽

Two Phase ◽

Radiographic Severity ◽

Cyclooxygenase 1 ◽

Prostaglandin Endoperoxide Synthase ◽

Top 40

ObjectiveTo identify genetic associations with severity of radiographic damage in ankylosing spondylitis (AS).MethodWe studied 1537 AS cases of European descent; all fulfilled the modified New York Criteria. Radiographic severity was assessed from digitised lateral radiographs of the cervical and lumbar spine using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). A two-phase genotyping design was used. In phase 1, 498 single nucleotide polymorphisms (SNPs) were genotyped in 688 cases; these were selected to capture >90% of the common haplotypic variation in the exons, exon–intron boundaries, and 5 kb flanking DNA in the 5′ and 3′ UTR of 74 genes involved in anabolic or catabolic bone pathways. In phase 2, 15 SNPs exhibiting p<0.05 were genotyped in a further cohort of 830 AS cases; results were analysed both separately and in combination with the discovery phase data. Association was tested by contingency tables after separating the samples into ‘mild’ and ‘severe’ groups, defined as the bottom and top 40% by mSASSS, adjusted for gender and disease duration.ResultsExperiment-wise association was observed with the SNP rs8092336 (combined OR 0.32, p=1.2×10−5), which lies within RANK (receptor activator of NFκB), a gene involved in osteoclastogenesis, and in the interaction between T cells and dendritic cells. Association was also found with the SNP rs1236913 in PTGS1 (prostaglandin-endoperoxide synthase 1, cyclooxygenase 1), giving an OR of 0.53 (p=2.6×10−3). There was no observed association between radiographic severity and HLA-B*27.ConclusionsThese findings support roles for bone resorption and prostaglandins pathways in the osteoproliferative changes in AS.

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Fine-mapping cellular QTLs with RASQUAL and ATAC-seq

10.1101/018788 ◽

2015 ◽

Cited By ~ 3

Author(s):

Natsuhiko Kumasaka ◽

Andrew Knights ◽

Daniel Gaffney

Keyword(s):

Fine Mapping ◽

Population Level ◽

Regulatory Elements ◽

Causal Variant ◽

European Population ◽

Open Chromatin ◽

Gene Promoters ◽

Genetic Associations ◽

Functional Interpretation ◽

Allele Specific

When cellular traits are measured using high-throughput DNA sequencing quantitative trait loci (QTLs) manifest at two levels: population level differences between individuals and allelic differences between cis-haplotypes within individuals. We present RASQUAL (Robust Allele Specific QUAntitation and quality controL), a novel statistical approach for association mapping that integrates genetic effects and robust modelling of biases in next generation sequencing (NGS) data within a single, probabilistic framework. RASQUAL substantially improves causal variant localisation and sensitivity of association detection over existing methods in RNA-seq, DNaseI-seq and ChIP-seq data. We illustrate how RASQUAL can be used to maximise association detection by generating the first map of chromatin accessibility QTLs (caQTLs) in a European population using ATAC-seq. Despite a modest sample size, we identified 2,706 independent caQTLs (FDR 10%) and illustrate how RASQUAL's improved causal variant localisation provides powerful information for fine-mapping disease-associated variants. We also map “multipeak” caQTLs, identical genetic associations found across multiple, independent open chromatin regions and illustrate how genetic signals in ATAC-seq data can be used to link distal regulatory elements with gene promoters. Our results highlight how joint modelling of population and allele-specific genetic signals can improve functional interpretation of noncoding variation.

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A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos

Development ◽

10.1242/dev.113.4.1345 ◽

1991 ◽

Vol 113 (4) ◽

pp. 1345-1355 ◽

Cited By ~ 1

Author(s):

M. Xiang ◽

S.Y. Lu ◽

M. Musso ◽

G. Karsenty ◽

W.H. Klein

Keyword(s):

Binding Site ◽

Regulatory Element ◽

Regulatory Elements ◽

Gene Promoters ◽

Mobility Shift ◽

Specific Expression ◽

Lytechinus Pictus ◽

Nuclear Extracts ◽

Nuclear Factors ◽

Electrophoretic Mobility Shift Assays

The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at −70 bp to −75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at −721 bp to −726 bp. The proximal ‘G-string’ element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.

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BarkBase: Epigenomic Annotation of Canine Genomes

Genes ◽

10.3390/genes10060433 ◽

2019 ◽

Vol 10 (6) ◽

pp. 433 ◽

Cited By ~ 5

Author(s):

Kate Megquier ◽

Diane P. Genereux ◽

Jessica Hekman ◽

Ross Swofford ◽

Jason Turner-Maier ◽

...

Keyword(s):

Regulatory Elements ◽

Open Chromatin ◽

Genetic Associations ◽

Functional Studies ◽

Genomic Annotation ◽

File Formats ◽

Causal Variants ◽

Health And Disease ◽

Differential Gene ◽

Accessible Chromatin

Dogs are an unparalleled natural model for investigating the genetics of health and disease, particularly for complex diseases like cancer. Comprehensive genomic annotation of regulatory elements active in healthy canine tissues is crucial both for identifying candidate causal variants and for designing functional studies needed to translate genetic associations into disease insight. Currently, canine geneticists rely primarily on annotations of the human or mouse genome that have been remapped to dog, an approach that misses dog-specific features. Here, we describe BarkBase, a canine epigenomic resource available at barkbase.org. BarkBase hosts data for 27 adult tissue types, with biological replicates, and for one sample of up to five tissues sampled at each of four carefully staged embryonic time points. RNA sequencing is complemented with whole genome sequencing and with assay for transposase-accessible chromatin using sequencing (ATAC-seq), which identifies open chromatin regions. By including replicates, we can more confidently discern tissue-specific transcripts and assess differential gene expression between tissues and timepoints. By offering data in easy-to-use file formats, through a visual browser modeled on similar genomic resources for human, BarkBase introduces a powerful new resource to support comparative studies in dogs and humans.

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Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2002-2011.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2002-2011

Author(s):

M Nishizawa ◽

S Nagata

Keyword(s):

Binding Site ◽

Dna Sequence ◽

Regulatory Elements ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Nf Kappa B ◽

Nuclear Extracts ◽

Bacterial Endotoxins ◽

Responsive Element ◽

Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.

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Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2002 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2002-2011 ◽

Cited By ~ 84

Author(s):

M Nishizawa ◽

S Nagata

Keyword(s):

Binding Site ◽

Dna Sequence ◽

Regulatory Elements ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Nf Kappa B ◽

Nuclear Extracts ◽

Bacterial Endotoxins ◽

Responsive Element ◽

Stimulating Factor

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High resolution genetic mapping of causal regulatory interactions in the human genome

10.1101/227389 ◽

2017 ◽

Cited By ~ 1

Author(s):

Natsuhiko Kumasaka ◽

Andrew Knights ◽

Daniel Gaffney

Keyword(s):

Dimensional Space ◽

Regulatory Elements ◽

Ctcf Binding ◽

Mendelian Randomisation ◽

Common Disease ◽

Binding Motif ◽

Open Chromatin ◽

Physical Interaction ◽

Model Framework ◽

Data Set

AbstractPhysical interaction of distal regulatory elements in three-dimensional space poses a significant challenge for studies of common disease, because noncoding risk variants may be substantial distances from the genes they regulate. Experimental methods to capture these interactions, such as chromosome conformation capture (CCC), usually cannot assign causal direction of effect between regulatory elements, an important component of disease fine-mapping. Here, we developed a statistical model that uses Mendelian Randomisation within a Bayesian hierarchical model framework, and applied it to a novel ATAC-seq data from 100 individuals mapping over 15,000 putatively causal interactions between distal regions of open chromatin. Strikingly, the majority (>60%) of interactions we detected were over distances of <20Kb, a range where CCC-based methods perform poorly. Because we can infer the direction of causal interactions, the model also significantly improves our ability to fine-map: when we applied it to an eQTL data set we reduced the number of variants in the 90% credible set size by half. We experimentally validate one of our associations using CRISPR engineering of the BLK/FAM167A locus, which is associated with risk for a range of autoimmune diseases and show that the causal variant is likely to be a non-coding insertion within a CTCF binding motif. Our study suggests that many regulatory variants will be challenging to map to their cognate genes using CCC-based techniques, but association genetics of chromatin state can provide a powerful complement to these approaches.

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LincSNP 3.0: an updated database for linking functional variants to human long non-coding RNAs, circular RNAs and their regulatory elements

Nucleic Acids Research ◽

10.1093/nar/gkaa1037 ◽

2020 ◽

Vol 49 (D1) ◽

pp. D1244-D1250 ◽

Cited By ~ 1

Author(s):

Yue Gao ◽

Xin Li ◽

Shipeng Shang ◽

Shuang Guo ◽

Peng Wang ◽

...

Keyword(s):

Rna Editing ◽

Somatic Mutations ◽

Regulatory Elements ◽

Circular Rnas ◽

Open Chromatin ◽

Nucleotide Polymorphisms ◽

Functional Variants ◽

Hypersensitive Sites ◽

Topologically Associated Domains ◽

Non Coding Rnas

Abstract We describe an updated comprehensive database, LincSNP 3.0 (http://bioinfo.hrbmu.edu.cn/LincSNP), which aims to document and annotate disease or phenotype-associated variants in human long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) or their regulatory elements. LincSNP 3.0 has updated with several novel features, including (i) more types of variants including single nucleotide polymorphisms (SNPs), linkage disequilibrium SNPs (LD SNPs), somatic mutations and RNA editing sites have been expanded; (ii) more regulatory elements including transcription factor binding sites (TFBSs), enhancers, DNase I hypersensitive sites (DHSs), topologically associated domains (TADs), footprintss, methylations and open chromatin regions have been added; (iii) the associations among circRNAs, regulatory elements and variants have been identified; (iv) more experimentally supported variant-lncRNA/circRNA-disease/phenotype associations have been manually collected; (v) the sources of lncRNAs, circRNAs, SNPs, somatic mutations and RNA editing sites have been updated. Moreover, four flexible online tools including Genome Browser, Variant Mapper, Circos Plotter and Functional Annotation have been developed to retrieve, visualize and analyze the data. Collectively, LincSNP 3.0 provides associations among functional variants, regulatory elements, lncRNAs and circRNAs in diseases. It will serve as an important and continually updated resource for investigating functions and mechanisms of lncRNAs and circRNAs in diseases.

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