1. Presentation by: Hannah Mays UCF - BSC 4434 Professor Xiaoman Li
A Genetic Variant Associated With Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression
Presentation by: Hannah Mays
UCF - BSC 4434
Professor Xiaoman Li

2. Overview
Integration of genetic fine mapping, epigenetic profiling, and CRISPR/Cas9 genome editing
Focus on EDN1 gene
Protein product is ET-1, 21 amino acid peptide
Most potent, longest lasting, vasoconstrictor in humans
Promotion of atherosclerotic plaque development

3. Vascular DISEASE Abnormal condition of blood vessels
Coronary artery disease (CAD)
Leading worldwide cause of morbidity/mortality
Heritable
Other diseases:
Migraine headaches
Cervical artery dissection
Fibromuscular dysplasia
Hypertension

4. Genome wide association study (GWAS)
Genetic variants between people with and without condition associated with disease
PHACTR1 locus on chromosome 6, position 24
Within gene phosphatase and actin regulatory protein 1
SNP in third intron: rs
Locus Zoom Plot
Association of 1.81x10-42
Figure 1

5. Regulatory region discovery
Use of ENCODE project tissue samples
ENCODE phase 3 inclusion of four aortic artery samples
2 Homozygous major, 1 Heterozygous, 1 Homozygous recessive
Strong enhancer signal overlying rs
All demonstrate robust H3K27 Acetylation
Suggestion of existence of gene regulatory function in this locus in vascular tissue
H3K27 – 27 aa in H3 of histone, is a lysine – associated with active transcription

6. Figure 2

7. Targeted Deletion of SNP using Crispr
At rs in 6p24
Deletion of 88-bp region
Induced pluripotent stem cell line using CRISPR/Cas9
Two vascular cell types: Endothelial Cells and Vascular Smooth Muscle Cells
Tested for nuclease activity
50-fold higher expression in Endothelial Cells of EDN1

8. Crispr-cas9 system
Figure 4

9. Validation of regulatory effect on EDN1 expression
Determine if effect on EDN1 gene expression is specifically mediated by a genotype at rs
Generation of isogenic stem cell lines, either homozygous major or minor
Three clones of each genotype selected (A, G)
RNAseq conducted on each of the six samples to identify cis- and trans- regulatory effects of rs
Differentially expressed genes
Blood vessel development
Vasculature development
Blood vessel morphogenesis

10. Figure 4

11. Interaction of distal regulatory element and EDN1
How distal regulatory element rs affects EDN1 expression
One possibility – through direct contact with EDN1 promoter site
Found to be unlikely
How to measure three-dimensional chromatin topology?
Circularized chromosome conformation capture sequencing (4C-seq)
Performed in human coronary artery endothelial cells

12. Analysis of the Possibility of Direct Contact
4C-Seq Analysis
Low contact probability and no evidence for a focal loop between rs and EDN1 promoter
Possible intergenic area of overlap intergenic to EDN1 and PHACTR1
Area contains high levels of H3K27Ac signal in endothelial cells
Consistent with a superenhancer
Figure 5

13. Results of Chip-seq indicate possibility of a super-enhancer
Large regions of cis-regulatory DNA
Span up to 12.5 kb
Accumulate large quantities of coactivator proteins
Control cell-type specific gene expression programs
4 distinct peaks of H3K27Ac signal in common contact region
Figure 5

14. Direct effect SNP on vascular diseases
Increased endothelial production of ET-1 and subsequent binding to ETA receptor promotes atherosclerosis
However, homozygous minor allele (G) at rs associated with decreased risk for migraines, cervical artery dissection, and fibromuscular dysplasia
Possibility of downstream effects of EDN1
G allele at rs results in higher EDN1 expression, increased ET-1 binding to ETa receptor, vasoconstriction, and therefore reduced risk of migraine headache
Figure 7

15. Challenges to application of variant and need for future studies
Modest effect each variant has on gene expression on non-coding variation
20% increase in EDN1 expression
Possibility of genetic or pharmacological factors leading to larger effects
Understanding the distal regulation mechanism
Regulation of gene 600 kb away
Mechanism of cis-regulatory effect not through typical enhancer-promoter contact
Common contact site in the middle of EDN1 and rs
Contains enhancers, H3K27Ac signal
Enhancers may interact with insulators, other enhancers, and promoters for distal genes

16. Reference
Gupta, Rajat M., Hadaya, Jospeh, Trehan, Aditi, …, Schnabel, Renate B., Bernstein, Bradley E., & Kathiresan, Sekar. (2017). A genetic variant associated with five vascular diseases is a distal regulator of endothelin-1 gene expression. Cell, 170,