Transcription Factors and Complex Disease Development Dr. Ines Pineda Torra
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1 1 Ines Pineda Torra, PhD UCL Division of Medicine University College London Outline 1. Transcription factors (TFs): What they are and what they do Why SNPs in TFs can play an important role in disease development 2. Identified transcription factor variants and impact on disease risk (CVD and Type 2 diabetes) Traditional approaches (gene candidate studies) PPARγ Contemporary approaches (genome wide association studies/ GWAS) TCF7L2 SORT1 3. Study of functionality of transcription factor mutations: Techniques used depending on where the mutation is localized: a) Coding region (change in protein sequence change in function?) 2 b) Promoter/regulatory region (changes in the TF levels?) Transcription: basic concepts General transcription factors (GTFs): form a preinitiation complex and recruit RNA Polymerase Cofactors: modulate the activity of a transcription factor Histone/Chromatin modifying enzymes: by changing the methylation/acetylation of histones or methylation of DNA mediate transit changes in chromatin accessibility Sequencespecific DNAbinding TFs: facilitate or prevent transcription initiation of specific promoters 3 RNA synthesis: creating a copy of RNA (ss) from a sequence of DNA (ds) 1
2 Transcription factors and biological processes Ensures correct of specific genes at a specific time in response to internal and external stimuli Over 1,400 known and predicted TF in humans (Messina, 2004; Vaquerizas, 2009) Summary of biological processes regulated by TFs Developmental process Cellular process Metabolic process Response to stimuli Immune system process Tissue and organ development Cell cycle progression Cell differentiation Signal transduction 4 Reproductive process Localization Transcription factors and disease Diseases arise from the dysregulation of transcription: In monogenic disorders, 164 TFs are responsible for 277 diseases or syndromes (Vaquerizas JM, Nature, 2009) TF are overrepresented among oncogenes (Furney SJ, BMC Genomics, 2006) 1/3 human developmental disorders attributed to dysfunctional TFs (Boyadjiev SA, Clin Genet, 2000) 5 Healthy Disease Health Correct mrna of target genes Correct of proteins Optimal regulation of pathways a) Mutation in the coding sequence of a TF Disease muttf with altered activity Abnormal mrna of target genes Abnormal of proteins Altered regulation of pathways b) Mutation in a TF binding sequence 6 Altered binding of TF to target gene Altered TF 2
3 Outline 1. Transcription factors (TFs): What they are and what they do Why SNPs in TFs can play an important role in disease development 2. Identified transcription factor variants and impact on disease risk (CVD and Type 2 diabetes) Traditional approaches (gene candidate studies) PPARγ Contemporary approaches (genome wide association studies/ GWAS) TCF7L2 SORT1 3. Study of functionality of transcription factor mutations: Techniques used depending on where the mutation is localized: a) Coding region (change in protein sequence > change in function?) 7 b) Promoter/regulatory region (changes in the TF levels?) Traditional approaches: PPARγ variant Research on pathophysiological pathways (cells, experimental models) Candidate gene genetic studies Identification of variants associated with disease risk Genome wide/systems biology approaches: TCF7L2, SORT1 variants Research on pathophysiological pathways (cells, experimental models) Identification of novel pathways Identification of variants associated with disease risk 8 hpparα PPAR subfamily DBD LBD Targets of fibrates and peroxisome proliferators (rodents) Role in energy homeostasis and FA oxidation, inflammation hpparβ % 70% Role in energy homeostasis and inflammation hpparγ 1 hpparγ % 68% Control of adipocyte differentiation, insulin sensitivity and inflammation 3
4 Positive gene regulation by PPARγ Endogenous ligands Polyunsaturated fatty acids Eicosanoids Synthetic ligands: Glitazones Rosiglitazone (insulin sensitizer) 9,10dihydro15deoxy 12,14Prostaglandin J2 Retinoids (9cRA) PPAR RXR Pol II Target genes Cytoplasm 10 PPRE Nucleus Corepressor complex PPAR RXR Pol II Ligands PPRE PPAR RXR Corepressor complex Cofactor receptor dynamic cycle PPAR RXR Degradation Coactivator complex Coactivator complex PPAR RXR Pol II Target gene transcription 11 PPRE PPARγ and metabolic homeostasis Key regulator of adipocyte differentiation Regulates the of genes encoding enzymes controlling: Glyceroneogenesis Lipid storage Lipid synthesis Lipid uptake Lipolysis Central role in endocrine function of adipose tissue: adiponectin PPAR RXR Pol II PPAR target gene 12 PPRE 4
5 Missense mutations in human PPARγ C142R C190S C190W F388L D424N Pro12Ala P113Q C159Y R194W V318M R425C P495L hpparγ2 DBD LBD Pro12Ala is the most frequent variant for PPARγ (225%) Association studies examining the link between this variant and insulin resistance are contradictory Two large metaanalyses showed modest but significant increase in Type 2 diabetes risk for the Pro allele carriers (Altshuler, 2000; Ludovico, 2007) High frequency variant: modest effect=large subpopulation at increased risk for T2D Disease risk confirmed in GWAS (Zeggini, 2008; Saxena, 2007; Scott, 2007; Sladek, 2007; Steinthorsdottir, 2007; Tonjes, 2006) Pro12Ala genotype sensitive to environmental inputs: Ala allele is beneficial in lean subjects but may be detrimental when coexisting with obesity (Ek, 1999) 13 Transcription factor variants in coding regions: In vitro functional studies 14 Function Transcriptional activity DNA binding Coactivator recruitment/ Protein interactions Subcellular localization Phosphorylation status Assay Luciferase reporter assay Gel shift assay Chromatin immunoprecipitation (ChIP) GSTpull down (direct interaction) Coimmunoprecipitation (direct/indirect) ChIP (direct/indirect) Subfraction immunoblotting Immunofluorescence Immunoblotting How to test the transcriptional activity of a TF in vitro: Luciferase reporter assays Pol TF II Promoter Reporter gene Luciferase *mut Pol TF II Promoter Reporter gene Luciferase mrna mrna Reporter protein Light Light 15 Detection by a luminometer Detection by a luminometer 5
6 How to test the DNA binding activity of a TF in vitro: Gel shift assays Protein (TF) Specific competitor (unlabeled probe) Mutant competitor (mut unlabeled) Probe Antibody (against TF) Supershift DNA:TF:antibody Shifted DNA:TF complex Gel separation of complexes 16 Free DNA (labeled probe) Detection of labeled probe Pro12Ala PPARγ: Functional studies In vitro Modest reduction of target gene transactivation due to reduced DNA binding (Ala) (Deeb et al., Nat genetics 1998) Reduced ability to induce adipocyte differentiation (Ala) (Masugi et al., BBRC 2008) c Light units PPARγ2Pro PPARγ2Ala d 0 40 CV1 HepG2 3T3L1 Relative affinities of the Pro and Ala PPARγ2 isoforms to PPRE by gel shift analysis Light units Deeb et al., Nat genetics 1998 J3TKLuc ACOTKLuc LPL prom. Transactivation activity of the Pro and Ala PPARγ2 isoforms in transient transfection assays Functional studies for other PPARγ variants Lossoffunction mutations associated with familial partial lipodystrophy type 3 (Agostini, Cell Metab 2006; Ludtke, J Med Genet 2007) Mutations involve Cys in the DBD: C142R, C159Y, C190W and C190S C142R, C159Y, C190W are transcriptionally inactive %wild type maximum 18 Agostini,Cell Metab 2006 C142R C159Y C190W 6
7 Functional studies for other PPARγ variants (2) C142R, C159Y, C190W are unable to bind to DNA Mutants are located in the nucleus and interact with cofactors Mutants show dominantnegative activity on the wt receptor RXR Gene PPRE 19 modified from Agostini, Cell Metab 2006 Functional studies for other PPARγ variants (3) Mutants show dominantnegative activity on the wt receptor No virus WT 20 modified from Agostini,Cell Metab 2006 GFP C114R C142R Functional studies for PPARγ variants: in vitro Pro12Ala (Nterminal domain) Cys142Arg Cys159Tyr Cys190Trp DNA binding Transcriptional activity Adipocyte differentiation 21 (DNAbinding domain) 7
8 Pro12Ala PPARγ: in vivo functional studies Generation of a knockin mutant (PPARγ Ala/Ala ), (Heikkinen, Cell Metabolism 2009) PPARγ Ala/Ala mice are leaner, improved insulin sensitivity and plasma lipid profiles, longer lifespan Beneficial effects eliminated by high fat feeding Induced adiponectin signaling Changes in target gene (less than 25%) Altered recruitment of cofactors Increased risk of type 2 diabetes for pro allele carriers 22 Health Correct mrna of target genes Correct of proteins Optimal regulation of pathways a) Mutation in the coding sequence of a TF Disease muttf with altered activity Abnormal mrna of target genes Abnormal of proteins Altered regulation of pathways b) Mutation in a TF binding sequence 23 Altered binding of TF to target gene Variation in noncoding DNA Important functional properties in the noncoding region of the human genome GWAS revealed a large number of noncoding sequence variants associated with human diseases Susceptibility regions do not overlap proteincoding genes 24 Modified from 8
9 Variation in distantacting enhancers Noncoding regions may contain enhancers: gene regulatory sequences that act over long distances Can modulate gene independently of their orientation Enhancers can be located upstream, downstream or within their target gene Are frequently conserved across species and are enriched in specific marks (H3K4me1, p300 coactivator binding) H3K4me1 H3K4me3 25 Identification of distantacting enhancers Comparative genomics: sequence comparison between relatively close species Genomicwide maps of enhancer marks (ChIPchip, ChIPseq): Histone methylation signatures (H3K4me1) Transcriptional coactivator presence (p300) ChIP: Crosslink DNA to protein Shear DNA and immunoprecipitate protein Reverse crosslinks 26 Isolate DNA enriched for binding sites Genome wide maps of enhancer marks by ChIPchip and ChIPseq ChIPchip Hybridize to microarray ChIPseq Sequence and map to reference genome 27 Readout: hybridization intensities Readout: Coverage by extended sequence tags 9
10 Validation of enhancer activity in vitro: Luciferase reporter assays TF Enhancer Pol II Core promoter Luciferase TF Enhancer *mut Pol II Core promoter Luciferase mrna mrna Reporter protein 28 Light Enhancer activity Light Enhancer activity Validation of enhancer activity in vivo Transgenic mouse reporter assay TF LcZ gene Enhancer Minimal/Core promoter 29 Candidate enhancer amplified by PCR Cloned into Lac Z reporter gene Generation of transgenic mouse Tissue of interest stained for Lac Z activity (blue) Genome wide/systems biology approaches: TCF7L2, SORT1 variants Research on pathophysiological pathways (cells, experimental models) Identification of novel pathways Identification of variants associated with disease risk 30 10
11 31 Genomic variants, TCF7L2 and diabetes risk Type 2 Diabetes (T2D) is characterized by impaired insulin secretion and insulin actions Inability of βcells to increase insulin secretion to compensate for the reduced sensitivity to insulin Genetic variants associated with T2D identified through GWAS (Voight BF, 2010) Mechanisms by which they increase disease risk are not entirely known Many of the variants locate in noncoding regions and some affect transcription factors TCF7L2: transcription factor 7like 2 (previously known as TCF4) TCF7L2 variants predispose to type 2 diabetes Canonical Wnt signalling pathway Interaction of the βcatenin pathway and TCF7L2 in pancreatic cells 32 TCF7L2 and diabetes risk Genetic variants (rs and rs ) are associated with severe diabetic phenotypes: Reduced insulin secretion Reduced βcell function Loss of GLP1 mediated increase in insulin secretion rs T allele consistently associated with diabetes in different populations In nondiabetics rs T allele also associated with coronary artery disease and cardiovascular events 33 What are the molecular mechanisms? 11
12 TCF7L2 variation in noncoding regions Both TCF7L2 variants are located in putative introns No coding variant in linkage disequilibrium with rs has been identified What effect does the mutation have on: TCF7L2 levels? TCF7L2 function? Alternative splicing involved? Less active alternative spliced transcripts TCF7L2 Controversy on the effect of rs on TCF7L2 Are variants located in distantacting enhancers that affect TCFL2? 34 Identification of enhancers in human pancreatic islets Isolation of active regulatory elements: FAIRE FAIRE:Formaldehydeassisted isolation of regulatory elements (Giresi PG, 2007) Aim: Identification of genomic regions associated with regulatory activities by isolating nucleosomedepleted DNA fragments from chromatin ( open chromatin ) DNA segments that regulate transcription in vivo are characterized by the eviction of nucleosomes from chromatin Traditionally identified by their hypersensitivity to nucleases Nucleosomedepleted DNA: DNAse I hypersensitive sites Transcriptional start sites Active promoters 35 Nucleosomefree chromatin FAIREseq in human pancreatic cells Examined DNA from human islets in nondiabetics to identify chromatinfree sites encompassing regulatory elements 80,0000 open chromatin sites: Crosslink chromatin with formaldehyde 45% isletspecific 3,348 clusters of isletspecific open chromatin sites Shear by Extract with sonication phenolchloroform Perform nextgeneration sequencing of extracted fragments Most clusters located within 10kb of the TSS 38 SNPs at 10 T2D loci overlapped with isletspecific open chromatin sites: rs in TCF7L2 (C>T) In islet samples from individuals heterozygous for the T risk allele: T allele more abundant than C allele Allelic imbalance T allele may exert its effect by altering the accessibility of the TCF7L2 DNA 36 Gaulton KJ, Nat Genet
13 Enhancer activity of the risk allele Relative luciferase activity, MIN6 cells (fold change) Relative luciferase activity, 832/13 cells (fold change) Nonrisk (m) Nonrisk Risk Control vector C T Control vector C T Gaulton KJ, Nat Genet Stitzel, Cell Metab The T risk allele shows greater enhancer activity than the C allele in both orientations in two βcell lines 37 Sequence variation in TCF7L2 affects T2D susceptibility by altering its gene regulation and local chromatin structure in islet cells TCF7L2 variant and chromatin accessibility The T allele may exert its effect on T2D risk by altering the accessibility of the TCF7L2 DNA TCF7L2 risk allele Higher TCF7L2 38 modified from Groop L, Nat. Genet Increased risk of T2D may be associated with over of TCF7L2 TCF7L2 variant and diabetes risk in animal models Hypothesis: non coding SNPs in TCF7L2 affect T2D risk by modifying TCF7L2 levels Direct connection between variation in regulatory sequences and disease risk Cloning of human sequences spanning non coding DNA in the TCF7L2 gene The region in TCF7L2 gene associated with T2D risk harbors enhancers that drive the of the gene in vivo 39 13
14 TCF7L2 variant and diabetes risk in animal models (2) TCF7L2 deficient mice (/) & (/) Reciprocal phenotypes Mice overexpressing *TCF7L2 Low levels of functional TCF7L2 Higher levels of TCF7L2 Low levels of blood glucose and plasma insulin Glucose Tolerance Diabetes risk High levels of fasting insulin 40 *TCF7L2: mouse cdna/human enhancer sequences TCF7L2 and diabetes risk Region with SNP associated with diabetes risk TCF7L2 deficiency and over in mice Knock in C T mutation in TCF7L2 in mice Enhancer activity in vitro / in vivo Changes in TCF7L2? 41 Effects on glucose metabolism Genomewide approaches: Sort1 Genomewide association studies identified a 1p13 risk locus for dyslipidemia and myocardial infarction (Samani, 2007; Kathiresan, 2009; Teslovich, 2010) Homozygosity for the major alleles is associated with a 2040% increase risk of MI and 16mg/dl higher LDLC Major alleles at the locus are present in 6580% of whites Relevant SNPs are localized in a gene cluster: CELSR2, PSRC1, MYBPHL and SORT1 42 Musunuru et al., Nature
15 Sort1 and lipoprotein metabolism One study implicates SORT1 as the causative gene Musunuru et al. show that the SNPs more strongly associated with LDLC levels are localized to an intergenic region between CELSR2 and PSRC1 Lower LDLC levels associated with higher SORT1 and PSRC1 mrna levels Only SORT1 over results in decreased LDLC Reduced of SORT1 in liver leads to increased LDLC 43 Musunuru et al., Nature 2010 Sort1 and lipoprotein metabolism (2) The minor allele SNP with strongest association creates a functional C/EBPα site 44 Sort1 and lipoprotein metabolism (3) A second study shows opposite effects of Sort1 and LDLC levels (Kjolby et al., Cell Metab. 2010) SORT1 Hepatic release of lipoproteins Plasma LDLC levels 45 Atherosclerosis in LDLRKO mice 15
16 Traditional approaches: PPARγ variant Research on pathophysiological pathways (cells, experimental models) Candidate gene genetic studies Identification of variants associated with disease risk Genome wide/systems biology approaches: TCF7L2, SORT1 variants Research on pathophysiological pathways (cells, experimental models) Identification of novel pathways Identification of variants associated with disease risk
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