Quatitative assessment of chromatin sturcture

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Learn the latest eqigenetic techniques including: discriminating epigenetically inactive chromatin from active chromatin, discriminating between aberrant and Monoallelic DNA methylation, predicting gene expression levels via chromatin structure assay and analyzing how DNA methylation affects promoter activity.

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Quatitative assessment of chromatin sturcture

  1. 1. Quantitative Assessment of Chromatin Structure:Analysis of Epigenetically Regulated Gene PromotersFrancisco BizouarnGene Expression DivisionLife Science Group July 20, 2011Bio-Rad Laboratories
  2. 2. Structure of Chromatin Euchromatin (transcriptionally competent)EpiQ Chromatin Analysis Epigenetics: DNA methylation Histone modification Heterochromatin (transcriptionally silent)
  3. 3. EpiQ StrategyEpigenetically inactivated chromatin is inaccessible andprotected from nuclease digestion, it can be discriminatedfrom active chromatin using a nuclease accessibility assay
  4. 4. The Nuclease Accessibility AssayTraditional Protocol• Isolate nuclei from cells or tissue• Treat nuclei with a nuclease• Isolate genomic DNA (gDNA)• Assess digestion of selected gene regions by a) Southern analysis b) Real-time PCR c) Microarray
  5. 5. The Nuclease Accessibility AssayTraditional Protocol• Isolate nuclei from cells or tissue• Treat nuclei with a nuclease• Isolate genomic DNA (gDNA)• Assess digestion of selected gene regions by a) Southern analysis b) Real-time PCR c) MicroarrayEpiQ Chromatin Analysis• Simultaneously permeabilize cells and digest chromatin in situ• Isolate gDNA• Assess digestion of selected genes by real-time PCR
  6. 6. EpiQ Assessment of Chromatin Structure
  7. 7. EpiQ Workflow
  8. 8. Analysis of Target Genes
  9. 9. EpiQ VerificationCell Lines AnalyzedHela Cervical CancerPC3 Prostate CancerLNCaP Prostate CancerHCT15 Colon CancerGenes AnalyzedSilenced:RHO (reference)Constitutive:ACTB, B2M, GAPDH, TBPEpigenetic Regulated:ABCB1, CDH1, CDH13, p14ARF,p16INK4a, DAPK1, DKK1, GSTP1,PTGS2, TP73, WT1
  10. 10. Analysis of Target Genes: ACTB Hela RNA PC3 LN H15Hela PC399% Accessible 99% AccessibleLNCaP HCT1596% Accessible 98% Accessible
  11. 11. Analysis of Constitutive Target Genes
  12. 12. Analysis of Target Genes: CDH1 Hela RNA PC3 LN H15 Hela PC325% Accessible 69% AccessibleLNCaP HCT1595% Accessible 99% Accessible
  13. 13. Analysis of Epigenetic Target Genes (1) B
  14. 14. Analysis of Epigenetic Target Genes (2)
  15. 15. Analysis of Epigenetic Target Genes
  16. 16. Conclusions• We observe a good correlation between gene expression and promoter chromatin structure in genes that are epigenetically regulated in human cancer• A small change in chromatin structure results in a large change in mRNA expression (10% change = 3 to 4 fold change)• Intermediate states of chromatin accessibility are real and act to fine-tune gene expression levels• We conclude that EpiQ chromatin analysis provides useful information regarding epigenetic gene regulation
  17. 17. EpiQ Assay Application Analysis of an Outlier
  18. 18. Analysis of an Outlier
  19. 19. EpiQ Analysis of GSTP1 Hela 100% RNA PC3 50% RNA LN <1% RNA Hela PC3 LNCaP98% Accessible 50% Accessible 1% Accessible
  20. 20. Melt Curve Assay for GSTP1 Promoter MethylationMethod:• Perform bisulfite conversion of genomic DNA samples• Amplify GSTP1 promoter from bisulfite converted DNA and assess amplicon melt profileAfter bisulfite conversion:• Methylated C remains as C• Unmethylated C is converted to U T• Melt profile of methylated DNA has a relatively high Tm• Melt profile of unmethylated DNA has a relatively low Tm
  21. 21. DNA Methylation Analysis of GSTP1 Hela 100% RNA Findings: In PC3 cells the GSTP1 PC3 50% RNA promoter exists in two distinct states: LN <1% RNA 1. Unmethylated and accessible and 2. Methylated and inaccessibleHela Inaccessible LNCaP Meth PC3 Unmeth Meth Unmeth PC3
  22. 22. DNA Methylation Analysis of GSTP1 in PC3 Clones PC3 Clone 1 Clone 2 Clone 3Clone 4 Clone 5 Clone 6 Clone 7Clone 8 Clone 9 Clone 10 Clone 11
  23. 23. Analysis of an OutlierFindings: The GSTP1 promoter in PC3 cells exhibits monoallelic DNA methylation, a rare form of epigenetic regulation
  24. 24. ConclusionThe EpiQ Chromatin Assay can discriminate between twofundamentally different forms of epigenetic gene regulation:Gene inactivation by aberrant DNA methylation(cancer, stem cells, development)Monoallelic DNA methylation(imprinting, X-chromosome inactivation)
  25. 25. EpiQ Assay ApplicationChromatin Structure Adjacent to a Promoter
  26. 26. Chromatin Structure of CDH1
  27. 27. Model of Chromatin RegulationNaked DNA Promoter
  28. 28. Model of Chromatin RegulationExpressed Gene Promoter
  29. 29. Model of Chromatin RegulationPartially Expressed Gene Promoter
  30. 30. Model of Chromatin RegulationSilenced Gene Promoter
  31. 31. Conclusion• The chromatin structure of the proximal promoter correlates well with gene expression, whereas other upstream and downstream regions do not• This finding is consistent with recent findings regarding DNA methylation in promoters and gene bodies
  32. 32. Epigenetic Assay Comparison Chromatin Structure via EpiQ versusDNA Methylation via Melt-Curve Analysis
  33. 33. Chromatin Structure and Gene Expression Euchromatin (transcriptionally competent)EpiQ Chromatin Analysis Epigenetic Events: DNA methylation Histone modification Heterochromatin (transcriptionally silent)
  34. 34. Chromatin Structure vs DNA Methylation: ABCB1 Hela ABCB1 mRNA PC3 RNA Expression LN H15Chromatin Structure Hela PC3 LNCaP HCT15 79% Accessible 18% Accessible 15% Accessible 93% Accessible Hela U PC3 U LNCaP U HCT15 U M M M M Hela H15 PC3 LNCaPDNA Methylation
  35. 35. Chromatin Structure vs DNA Methylation: CDH1 Hela CDH1 mRNA PC3 RNA Expression LN H15Chromatin Structure Hela PC3 LNCaP HCT15 25% Accessible 69% Accessible 95% Accessible 99% Accessible Hela PC3 LNCaP HCT15 U U U U M M M M PC3 H15 Hela LNCaPDNA Methylation
  36. 36. Epigenetic Assay ComparisonConclusion:The EpiQ Chromatin Structure assay is better than a DNAmethylation assay in predicting gene expression levels
  37. 37. Epigenetic Assay ComparisonConclusion:The EpiQ Chromatin Structure assay is better than a DNAmethylation assay in predicting gene expression levelsImplication:Analysis of chromatin structure is more “biologically relevant”than analysis of DNA methylation
  38. 38. Types of Epigenetic Analyses EuchromatinEpiQ DNA methylation Histone modification Heterochromatin
  39. 39. Summary• We have developed a rapid and easy assay that assesses chromatin structure in cultured cells• This assay can complement and extend existing assays and provide new insights into epigenetic gene regulation• This assay can allow researchers to ask new questions and obtain new insights in several areas of biomedical relevance
  40. 40. AcknowledgementsSteven Okino Yan WangMichelle Kong Janice LewSherman Cheng Carlos Barraza Evan Bursey Adam McCoy Kamni Vijay Mark Chilcott Viresh Patel Xiao-Song Gong Liz Jordan Hannah Yuan Naheed Aslam Beth Ladin Scott Crowder Ramesh SathiyaaJimmie Lowery Gene Expression Division Life Science Group Bio-Rad Laboratories

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