Gene Regulation, Epigenetics & Databases
Cindy G Boer Genetic Laboratory
Internal Medicine
Erasmus MC
Congratulations!
A genome-wide significant GWAS hit!
(and what to do now?)
Annotation of genetic loci
Where is your SNP? & What could it do? 1. Coding or in non-coding DNA
2. In a gene body or in an intergenic region ?
3. In a regulatory region? – Promoters, enhancers, inhibitors, insulators, transcription factor binding
sites etc.
4. Causal gene & Mechanism?
Causal Variant, Causal Gene, Causal cell type
Linkage Disequilibrium (LD)
• Association between disease trait and (tag) SNP – Array designed on LD structure not functional SNP
• Identification of Causal variant?
• LD structure plotted • SNPs high LD • (r2 >0.8 or r2 > 0.6)
Castaño Betancourt, et al.,(2016), PLOS genetics
Genome-wide association signal (Best case scenario)
Top SNP (+SNPs LD >0.8) is located in the coding sequence of a gene
• Synonymous? Or Non-Synonymous?
• Gene? What is known, what does it do? – Damaging effect of the hit?
(first part of the practical)
Genome-wide association signal (Realistic scenario)
Most GWAS findings are located in non-coding regions of the genome [M.T. Maurano et al., Science, 337, 1190 (2012)]
– Introns or intergenic
– ~ 98.5% human genome is non-coding
Difficult to link SNP phenotype
Regulatory elements
GWAS SNPs are enriched for regulatory elements.
Regulatory regions Promoters, enhancers, inhibitors, insulators, transcription factor binding sites etc.
1. What is a regulatory region/how is a regulatory region defined?
2. How will you know if your hit is located in a regulatory region?
[M.T. Maurano et al., Science, 337, 1190 (2012)]
Gene Expression
• Promoter : region of DNA that initiates transcription of a gene
• Enhancer : short region of DNA that increases/helps initiate the transcription of a gene.
• Inhibitor : short region of DNA that decreases/inhibits the transcription of a gene.
• The regulation/control of gene expression is essential for cell function, survival, differentiation etc.
Epigenetics = Changes/regulation of gene expression, caused by mechanisms other than DNA sequence variation
Enhancer
The Central Dogma (of molecular biology)
Epigenomics:
All epigenetic modifications on the genetic material of a cell
The Central Dogma
Animals: ~100-150 different cell types
“Same Blueprint of DNA each cell”
How are there different cell types?
Epigenetics
Epigenetics
“The study of changes in
gene expression or cellular
phenotype, caused by
mechanisms other than
changes in the underlying
DNA sequence”
“Epigenetic mechanisms can
control the functions of
noncoding sequences of
DNA”.
Epigenetics
Histones & Chromatin
Histones & histone modifications
DNA structure & Regulation
DNase hypersensitive regions open chromatin configuration
DNA structure & Regulation
The Histone Code
Histone code: multiple histone modifications specific unique downstream functions
Specific proteins involved in gene control recognize and interrogate the patterns of histone modifications: Ex. RNA polymerase II, Transcription factors & DNA binding proteins
– Transcription factor recruitment
– Chromatin shape and function
Epigenetics: Histone Code
Inactive Promoter Active Promoter
H3K27me3 H3K4me3 [promoter specific]
DNA methylation H2A.Z [histone variant]
Inactive Enhancer Active Enhancer
H3K9me2 H3K4me1 [enhancer specific]
DNA methylation H2A.Z [histone variant]
Many many (100+) different histone modifications known! very complex!
Regulatory regions: Chromatin States
ENCODE/ROADMAP • “15-state model” • Histone modifications • DNAse sites • TF-binding Sites
Roadmap Epigenomics Consortium, et al., Nature 2015
Epigenetics: symphony No. 9
DNA binding proteins
DNA-binding proteins: Transcription factors, nucleases, other DN binding proteins
Non-specific binding: polymerases, histones
Specific binding: Transcription factors, nucleases
Specific binding recognition consensus sequence
Change in consensus sequence change in DNA binding affinity? change in gene regulation/expression?
Consensus sequences
• DNA binding motif: “recognition sequence”
• Found in databases:
– JASPAR database
– Integrated in HaploReg (practical)
Can also be affected by methylation! (EWAS)
CTCF methylation
CTCF binding is affected by methylation in it’s core sequence
Proper CTCF functioning is essential!
“severe dysregulation of CTCF in cancer cells”
Mouse mutants CTCF – embryonic lethal
So Far we have:
Annotation: • Location (Chr/Bp)
• Coding/non-coding
• DNA regulatory elements – (and open chromatin sites)
• Transcription factor binding sites
GWAS & EWAS goal Identify novel targets/genes involved in phenotype X
So far only annotation, No (potential) causal gene
Gene Regulation
Adapted from: Alberts, Molecular Biology of the Cell 5th Edition, figure 7-44
Typical eukaryotic gene regulation • Complex 3D looping (CTCF) • Multiple regulatory regions • Involvement of multiple transcription factors • Can be cell type specific
Gene regulation is highly complex!
Gene Regulation
• ~1 MB (1000.0000 base pairs) long range regulation
– Sonic Hedgehog, essential developmental gene
Circadian rhythm : Epigenetics
• Mammalian circadian clock
• Oscillation of ~ 24h
– Light-dark cycle (melatonin secretion), Feed cycle
• A conserved transcriptional–translational auto-regulatory loop generates molecular oscillations of ‘clock genes’ at the cellular level
PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription, Zhao et al., 2015 Molecular Cell
Complex 3D structure
Finding [causal] Genes
Cell type specify is useful & Important:
• Gene expression levels (RNA-seq)
– Predicted promoter activity in cell type
– Predicted gene activity (ex active gene transcription mark: H3K36me3)
• Gene expression – Genotype
– eQTL’s! (Thursday lecture/practical)
– Also Cell type specific!
Causal Genes: Example
Enhancer site (likely) to regulate gene 1 or gene 2?
Cell type selection:
• Not in all cases the selection of target tissue will be easy: – Cell fate – Cell state and Cell type – Complex diseases & phenotypes
Availability of material & data Proxy tissues:
• Same lineage, similar functioning tissue • (gene of interest) expression vs no expression
• Tools & databases to select target tissue • GWAS SNPs are enriched for gene regulatory regions….in
target cell type!
Phenotype - Alzheimer
Enhancer Marks in Brain?
Enhancer Marks in Heart?
• Central Dogma: DNA- RNA-Protein & gene regulation is everything!
• DNA regulatory elements: promoter, enhancer, inhibitor
• Epigenetics is cell type specific, think on what cell type is relevant to you
Go and Annotate your GWAS hit
Genome-wide association signal
..How to Find?
• Where is your hit (SNP) located? – Chromosome & position – Near or in which genes
• Coding variant – Synonymous/non-synonymous
• Regulatory regions • 3D structure of the genome • Candidate gene
– gene function
• Cell type?
• Online collection of (molecular) biological data
– Structured & Searchable
– Publically available
– Updated periodically & Cross-referenced
• Literature
• Data from research
Biological databases
• Pubmed – Literature database • Categorized databases: to much to name
– Genomic variation: dbSNP, HapMa .... – Sequence: NCBI RefSeq database, Entrez Nucleotide, miRbase... – Proteins: RCSB protein databand, UniProt, SMART... – Pathways: KEGG, Reactome, STRING... – DNA annotation: ENCODE, ROADMAP epigenetics
• Genome Browsers: genomic database, integrating all data associated to genome annotation & function.
• Mining Tools: FUMA & HaploReg
Genome Browser
• Displaying, viewing and accessing genome annotation data
• Genome annotation:
– DNA-variation information, epigenetic regulation, transcription, translation, disease information...
• Links to other specialized Databases
Difference?
• NCBI, UCSC and EnsEMBL use the same human genome assembly generated by NCBI – Release timing and data availability can differ between sites
• NOTE: the version of the genome assembly – Annotation location and availability will be different between different
assemblies
• Own preference which to use
• Practical: mainly UCSC and some forays into other databases, including NCBI, EnsEMBL & ENCODE
Mining Tools
FUMA
Functional Mapping and Annotation of Genome-Wide Association Studies
– Monday Practical & Todays practical
– Novel Tool!
Mining Tools
HaploReg HaploReg is a tool for exploring annotations of the noncoding genome at variants on haplotype blocks, such as candidate regulatory SNPs at disease-associated loci.
• Mine ENCODE & RADMAP data be careful! Not always up to-date or gives clear information!
Your Research
Play with the tools
Lot’s of (useful) information
• Be critical
– Check the outcome
– Know the data
– References
– Hypothesis building only!
Go and get lost... (and write down where you went)
Your research NEEDS biological databases!
The Practical
• UCSC genome browser links to other databases & data – Ensembl, ENCODE, ROADMAP, HaploREG, FUMA, GTEX………..
• 3 part practical I. Beginner database and bioinformatictools (FUMA, UCSC, HaploReg)
II. Advanced: adding regulatory data and gene expression data
III. More Advanced: Adding 3D chromatin structure to your annotation
Focus on “real life” examples
Use for your own research!
UCSC Genome Browser
UCSC Genome Browser
UCSC Genome Browser
Hints for the Practical • Ask us anything (me, Linda & Joost)
- (related to the practical or genetics)
• DNA is LARGE and a 3D molecule
– So check your surroundings! (i.e. zoom out)
• Can I click on it? YES more information! more track control!
• GIYF: Google is your friend
• Practical is in 3 parts
– Intro – standard – difficult
& Enjoy (or try to)
Questions?
