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) • LD structure plotted • SNPs high LD • (r 2 >0.8 or r 2 > 0.6) • Association between disease trait and (tag) SNP – Array designed on LD structure not functional SNP • Identification of Causal variant? 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 Enhancer • 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
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” Epigenetics “Epigenetic mechanisms can control the functions of noncoding sequences of DNA”.
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 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! Adapted from: Alberts, Molecular Biology of the Cell 5 th Edition, figure 7-44
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 Enhancer Marks in Marks in Brain? Heart?
Genome-wide association signal • 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
..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!
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