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Getting to the heart of genomic dark matter

Getting to the heart of genomic dark matter Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart specific long non-coding RNAs. Samir Ounzain, Ph.D Pedrazzini Lab Department of Medicine Experimental Cardiology Unit

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Getting to the heart of genomic dark matter

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  1. Getting to the heart of genomic dark matter Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart specific long non-coding RNAs. Samir Ounzain, Ph.D Pedrazzini Lab Department of Medicine Experimental Cardiology Unit University of Lausanne Medical School Switzerland @ispiyou @CardiolncRNA Ounzain S, Micheletti R, Beckmann T, Schroen B, Alexanian M, Pezzuto I, Crippa S, Nemir M, Sarre A, Johnson R, Dauvillier J, Burdet F, Ibberson M, Guigó R, Xenarios I, Heymans S, Pedrazzini T. Genome-wideprofiling of the cardiactranscriptomeaftermyocardialinfarction identifies novelheart-specific long non-codingRNAs. EurHeart J. 2014 Apr 30.

  2. Response to biomechanical stress in the adult heart What we want: More myocytes Less fibrosis What we have: Fewer myocytes More fibrosis Biomechanical Stress Myocardial Infarction Hypertension Electro-mechanical Remodeling Normal Compensated Failing Cardiomyocyte hypertrophy Gradual loss of cardiomyocytes Fibroblast proliferation Massive loss of myocytes Fibrosis Regeneration Mobilization of immature cardiomyocytes and cardiac stem cells

  3. Cardiac gene regulatory networks

  4. Long non-coding RNAs – Characteristics and mechanisms of action Typically (A) >200bp (B) lack coding potential (C) PolyA(+) (D) Pol2 transcribed (E) multi-exonic Highly tissue/context specific – Thousands likely exist Hu, W et al (2012) Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO Reports Functions in the heart are poorly characterized

  5. Characterizing the mouse long non-coding (lncRNA) transcriptome 5.6% 8.7% (1521) 85.7% 14 d. Myocardial infarction Border zone Poly A(+) RNA-Sequencing UCSC mRNA UCSC lncRNA ab initio reconstruction Novel lncRNA Association with specific chromatin state transitions Correlation with cardiac physiology Heart specificity Functional roles as Enhancer derived ncRNAs p300 H3K4me1 H3K4me3 H3K27Ac H3K27me3 Mouse enhancer mRNAs Novel lncRNAs UCSC lncRNAs Human ortholog identification 1110 lncRNAs Human heart disease Mouse model LV LA RA RV

  6. Novel lncRNAs exhibit significant cardiac specific expression All transcripts Novlnc6 15075 UCSC mRNAs Heart specificity Heart specificity 988 UCSC lncRNAs Cufflinks Predictions + Heart - + Testis - + Heart specificity 1521 Novel lncRNAs Kidney Strand specific transcription - + Liver - + Lung - + Stomach Heart - Sham Heart MI Heart 17 non cardiac tissues PhastCons Vertebrates

  7. Novel lncRNAs exhibit significant cell and sub-cellular specificity Fibr. CM Novlnc333 Novlnc174 Fibr. CM Novlnc95 Novlnc86 Novlnc11 Novlnc44 Novlnc90 Novlnc95 Novlnc103 Novlnc49 Novlnc96 Novlnc49 Novlnc76 Novlnc11 Novlnc86 Novlnc15 Novlnc32 Novlnc44 Novlnc76 Novlnc96 Novlnc61 Novlnc6 Novlnc90 Novlnc35 Novlnc15 Novlnc23 Novlnc6 Novlnc35 -4 -2 2 4 -0.15 -0.10 -0.05 0.05 0.10 0.15 0 0 Novlnc333 Novlnc61 Novlnc174 Novlnc103 Novlnc32 Novlnc23 Cyto. Nuc. Delta Fold Change Cyto/Nuc Ratio

  8. Novel lncRNAs are highly correlated with cardiac physiological traits Echocardiography derived traits 1. 2. 3. Heart specific transcripts (%) Novel lncRNAs -1.0 -0.5 0 0.5 1.0 Correlation 4. EF FS IVRT IVS; s IVS; d LVID; s LVID; d LVPW; s LV mass LVPW; d Heart rate SA MI trace LA MI trace Body weight LV volume; s LV volume; d LA LV area; d LA LV area; s SA LV area; d LV mass / BW EF MI Trace 2.0 2.0 EF 5 Cluster 1 4 Cluster 2 1.5 1.5 Density 3 Cluster 3 2 Density Density Cluster 4 1 UCSC lncRNAs 1.0 1.0 0.25 0.50 0.75 1.00 Novel lncRNAs Correlation Correlation 0.5 0.5 0.25 0.50 0.75 1.00 0.25 0.50 0.75 1.00 Correlation

  9. Novel lncRNAs are highly correlated with cardiac physiological traits Border Zone Novlnc35 Novlnc174 Novlnc333 mhy7 Myocardial infarction col1a ctgf nppa tgfb2 Novlnc61 Novlnc23 Novlnc86 Novlnc11 R=-0.788 Novlnc44 Novlnc49 Novlnc95 Novlnc6 Novlnc15 Novlnc96 Border zone Novlnc76 R=-0.484 Novlnc90 Novlnc103 R=0.811 Novlnc32 EF FS IVS; s IVS; d Myh7 LVID; s LVID; d LV mass LVPW; s LVPW; d Heart rate LA MI trace SA MI trace Body weight LV volume; s LV volume; d LA LV area; s LA LV area; d SA LV area; d LV mass / BW R=-0.426 Novel lncRNAs Cardiac markers EF (%) R=-0.538 Novlnc95 Novlnc6 R=0.785 EF (%) Novlnc174 Fold change over Sham Fold change over Sham R=0.785 -0.5 0 0.5 Nppa 70 70 70 70 70 70 70 50 50 50 50 50 50 50 30 30 30 30 30 30 30 10 10 10 10 10 10 10 -3 -2 -1 0 -1 0 1 2 Novlnc15 Col1a -4 -3 -2 -1 0 -2 -1 0 1 2 -3 -2 -1 0 1 -1 0 1 2 3 1 0 1 2

  10. Novel lncRNAs are associated with cardiac specific active enhancers H3K4me3+H3K27me3bivalent/poised promoterH3K4me3+H3K27acactive/initiating promoterH3K4me3initiating promoterH3K27me3+H3K4me1poised developmental enhancerH3K4me1poised enhancerH3K27ac+H3K4me1    active enhancer H3K27me3Polycomb repressed ENCODE Data Sets ChIP-Seq data sets from 5 mouse whole tissues

  11. Novel lncRNAs are associated with cardiac specific active enhancers Novlnc6 Cufflinks Predictions Heart Heart Heart Heart Kidney Kidney Kidney Kidney H3K4me1 Liver Liver Liver Liver Spleen Spleen Spleen Spleen Testis Testis Testis Testis H3K4me3 H3K27Ac H3K27me3 PhastCons Vertebrates Enhancer

  12. Inferring functions for novel lncRNAs based on chromatin state transitions Stages of differentiation Cardiomyocytes Embryonic stem cells Mesodermal precursors Cardiac precursors Lineage-specific gene expression Expression Genes x y z x y z x y z x y z Based on Wamstad JA et al. 2012. Cell 151: 206 Paige SL et al. 2012. Cell 151: 221

  13. Inferring functions for novel lncRNAs based on chromatin state transitions Stages of differentiation Cardiomyocytes Embryonic stem cells Mesodermal precursors Cardiac precursors Lineage-specific gene expression Expression Genes x y z x y z x y z x y z Enrichment H3K4me3 Enrichment H3K27me3 Functional inference Chromatine state transition Enrichment H3K4me1 Enrichment H3K27Ac Based on Wamstad JA et al. 2012. Cell 151: 206 Paige SL et al. 2012. Cell 151: 221

  14. 1st pattern Cluster 1 (gene x, plus other genes with similar pattern) 2nd pattern Cluster 2 (gene y, plus other genes with similar pattern) 34 Patterns / Clusters 3rd pattern Cluster 3 (gene z, plus other genes with similar pattern) Wamstad JA et al. 2012. Cell 151: 206

  15. Novel lncRNAs are associated with cardiac specific chromatin state clusters Wamstad JA et al. 2012. Cell 151: 206 Definition of 34 clusters corresponding to 34 groups of genes that are functionally related E.g.: Cluster 1, 2, 3: House keeping function Cluster 18: Immune/Inflammatory response Cluster 20, 23-27: Cardiac development, muscle contraction Cluster 28: Calcium homeostasis, GPCR signaling USCS mRNAs Down Unchanged Up 27 28 25 26 1 1 23 24 20 2 Pearson residual 18 3 2 18 3 20 23 Novel lncRNAs 24 25 28 27 1 26 26 27 2 28 3 25 Sham vs. MI 24 Cluster-associated novel lncRNAs are enriched in –up/-down regulated lncRNAs 23 20 18 7.25 4.00 2.00 0.00 - 2.00 - 4.00 - 5.70

  16. Candidate cardiac enhancer derived lncRNA – Novlnc6 Novlnc6 Nppa Col1a EF * * % * Fold change over Sham d1 * d1 d1 d1 d1 d1 d7 d7 d7 d7 d7 Fold change over Sham d1 R=-0.788 * R=0.785 * * Border Zone Remote Zone Novel lncRNAs Chromatin State Clusters Novlnc6 28 27 1 26 Bmp10 2 25 3 24 Nkx2.5 23 Cardiomyocytes 20 18 * Novlnc6 Gapmers Novlnc6 Bmp10 Nkx2.5 Gata4 Tbx20 Myh6 Myh7 Scrambled * * Fold change over Scramble * * Novlnc6 Gapmer 1 ** ** Novlnc6 Gapmer 2 Novlnc6 Nppa 70 70 EF (%) 50 50 30 30 10 10 -3 -2 -1 0 -1 0 1 2 p300 H3K4me1 H3K4me3 H3K27Ac H3K27me3 PhastCons Vertebrates Mouse enhancer LV LA RA RV

  17. Novlnc6 is modulated in human heart disease Hs Novlnc6 TransMapped Human Orthologs 1100 human orthologs identified Mammalian Basewise PhyloP PhastCons Vertebrates Dilated cardiomyopathy Hs Novlnc6 Nkx2.5 Nppa Col1a EF PW thickness p < 0.001 p < 0.05 p < 0.001 p < 0.001 p < 0.001 Aortic stenosis p < 0.01 p < 0.01 p < 0.001

  18. Summary • Identified 1500 novel lncRNAs in the infarcted mouse heart • Novel lncRNAs are heart specific • Novel lncRNAs are highly correlated with physiological traits • Novel lncRNAs are enriched at heart specific active enhancers • Inferred novel lncRNA functions based on chromatin states • Conserved novel lncRNAs are modulated in human disease Unique characteristics of novel lncRNAs render them ideal tissue specific therapeutic targets and biomarker candidates

  19. Rudi Micheletti Tal Beckmann Michael Alexanian Iole Pezzuto Stefania Crippa Mohamed Nemir Pedrazzini Lab @ispiyou @CardiolncRNA Jerome Dauvillier Frederic Burdet Mark Ibberson Ioannis Xenarios Rory Johnson Roderic Guigo Blanche Schroen Stephane Heymans Alexandre Sarre Keith Harshman

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