Chromatin-Based Regulation of Gene Expression

Chromatin-Based Regulation of Gene Expression.George J. Quellhorst, Jr., PhD.Associate Director, R&D.Biological Content Development

Topics to be Discussed Importance of Chromatin-Based Regulation Mechanism & Pathway Reading, Writing, Erasing the Code Typical Questions Asked & Methods Used Examples from the Literature How QIAGEN can help - 2 -

Transcriptional Regulation of Gene Expression A Current Picture TF Activating Signal Transduction Cascades THE Gene Expression Regulation Frontier! Highly Dynamic Promoter Context p53 NFκB + p53 BS NFκB BS Distal Promoter Histones Me? Histone Modification Me SWI/SNF or INO80 or Mediator Complex Me Me TBP Me Me RNA Pol II TSS (+1) Me DNA Methylation Structural Gene mrna or mirna OAc OAc OAc Proximal Promoter - 4 -

Reading, Writing, Erasing DNA Methylation O N N NH 2 NH 2 NH H 2 N N + DNMT - DNMT O O CH 3 HCH3 N S -Enz N AdoMet AdoHcy R Cytosine 5-Methyl-Cytosine Modification of cytosine in CpG dinucleotide at 5-position with methyl (CH 3 ) group Written by DNA Methyltransferases DNMT1: Maintenance Methylation DNMT3A & DNMT3B: De Novo Methylation Read by Transcription Factors Methyl DNA Binding Protein Histone Modification Enzymes Erased by DNA Demethylases? Tumor Suppressor Genes Normal Promoter Region 1 2 3 Expression Cancer 1 2 3 No Expression Herman JG, Baylin SB. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 20:2042. 2003 Massachusetts Medical Society - 5 -

DNA Methylation Dependent Transcriptional Repression Possible Molecular Mechanisms Singal R and Ginder GD (1999) DNA Methylation. Blood 93:4059. 1999 by The American Society of Hematology - 6 -

Occurrence & Study of DNA Methylation.70% to 90% of CpG dinucleotides are methylated in healthy somatic cells Representing 3% to 6% of all cytosines Less than expected frequency based on genomic GC content.cpg Island = relatively low GC content but high CpG 7% of all CpG dinucleotides Associated with 5 regulatory regions of 40-60% of human genes Typically unmethylated.cancer Genome-wide hypomethylation except CpG islands ~ X chromosome inactivation.development Cell-type specific methylation @ proximal promoter of affected genes.5-azacytidine (5-aza-C, DAC) Keeps CpG from being methylated turning gene expression back on Cancer treatment Mechanism of action experimental tool - 7 -

The Histone Code Acetylation H3ac Active Euchromatin H3K9ac Active Euchromatin H4ac Active Euchromatin.Methylation.H3K4me1 Non-TSS.H3K4me2 Active Euchromatin.H3K4me3 Active Euchromatin.H3K9me1 Inactive Euchromatin.H3K9me2 Heterochromatin.H3K9me3 Heterochromatin.H3K27me3 Inactive Euchromatin.H3K36me3 Non-TSS.H3K79me3 Active Euchromatin.H4K20me3 Heterochromatin.Phosphorylation & Ubiquitination.Less clear Kondo Y. (2009) Epigenetic cross-talk between DNA methylation and histone modifications in human cancers. Yonsei Med J. 50:455. Yonsei University College of Medicine 2009-8 -

Reading, Writing, Erasing the Histone Code.Histone Lysine Acetylation Neutralizes basic histone charge preventing higher order compaction More open and accessible for transcription Written by Histone Acetyltransferases (HATs) Read by Bromodomain proteins Chromatin Remodeling Factors Erased by Histone Deacetylases (HDACs) Trichostatin A (TSA) inhibits most HDACs Class I (HDAC1-3, HDAC8) & Class II (HDAC4-7, HDAC9-10) Allows histones to be re-acetylated turning gene expression back on Cancer treatment Mechanism of action experimental tool.histone Lysine Methylation Closed form, inactive for transcription Written by Trithorax Group & SET domain proteins Read by ING family & Chromobox homolog proteins Chromatin Remodeling Factors Erased by Jumonji Domain (JARID/KMD) Lysine Demethylases - 9 -

DNA Methylation & Histone Modification Interplay Ikegami K, Ohgane J, Tanaka S, Yagi S, Shiota K. (2009) Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development. Int J Dev Biol. 2009;53(2-3):203-14. 2009 UBC Press - 10 -

DNA Methylation & Histone Modification Interplay Kondo Y. (2009) Epigenetic cross-talk between DNA methylation and histone modifications in human cancers. Yonsei Med J. 50:455. Yonsei University College of Medicine 2009-11 -

Examples of Experimental Studies Questions Asked & Methods Used.Question: Is expression of my gene of interest (GOI) regulated by epigenetics? Is its promoter methylated? What histone modifications localize at its transcription start site? Do these epigenetic marks interfere with transcription factor (TF) binding? Can its silenced expression be turned on again and TF binding be restored by erasing epigenetic marks?.methods: DNA Methylation Analysis: Bisulfite Treatment (Converts C but not Me-C to U) followed by Microarray, Sequencing Methyl-Specific PCR primers without Cs except at 3 -end Histone Modification and Transcription Factor (TF) Binding Analysis Chromatin Immunoprecipitation (ChIP) followed by PCR Anti-histone modification or anti-tf pull down & DNA purification PCR primers to amplify promoter region for GOI Erase epigenetic marks with 5-aza-C and/or TSA - 12 -

Promoter Methylation Decreases Gene Expression ERBB4 (HER4) in Breast Cancer Cell Lines Why are some breast cancers HER4 positive and some HER4 negative? RT-PCR: HER4 Bisulfite Pyrosequencing Results More Methylation Less Expression Das PM, Thor AD, Edgerton SM, Barry SK, Chen DF, Jones FE. (2010) Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells. Oncogene 29:5214. 2010 Macmillan Publishers Limited - 13 -

Methylation Inhibits TF Binding but Recruits MeCP2 CREB, ATF2 and MeCP2 Binding to Insulin Promoter What are the consequences of tissue-specific methylation of the insulin promoter? Chromatin Immunoprecipitation followed by PCR from NIT-1 mouse insulinoma cell line IP: Various TFs PCR: Endogenous Promoter IP: Various TFs PCR: Transfected Reporter Plasmids Kuroda A et al. (2009) Insulin gene expression is regulated by DNA methylation. PLoS One. 4:e6953. 2009-14 -

Is Increased Gene Expression & TF Binding Epigenetic? Ethanol & 5-aza-C Similarly Increase GRIN2B Expression & TF Binding RT-PCR: GRIN2B CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawal CIEW5: CIE + 5-day withdrawal 5 AZA: 5-Azacytidine Why does ethanol activate expression of GRIN2B? Chromatin Iummunoprecipitation IP: Various TFs PCR: GRIN2B Promoter TFBS Primary cortical cultured neurons Qiang M, Denny A, Chen J, Ticku MK, Yan B, Henderson G. (2010) The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS One 20:e8798. 2010-15 -

Removing Methylation Restores Gene Expression Ethanol Treatment Decreases GRIN2B Methylation, DNMT1 Expression Why does ethanol activate expression of GRIN2B? SAM = S-adenosyl-L-methionine, DNMT substrate Rescues hypomethylation RT-PCR: DNMT1 Bisulfite Pyrosequencing Results CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawal CIEW5: CIE + 5-day withdrawal 5 AZA: 5-Azacytidine Qiang M, Denny A, Chen J, Ticku MK, Yan B, Henderson G. (2010) The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS One 20:e8798. 2010-16 -

Epigenetic Regulation Keeps Oncogenes Suppressed MUC1 Expression & Regulation in Different Cancer Cell Lines Why do some cancers NOT express MUC1, a potential marker for malignancy? Cell Line PANC1 MDA-MB-453 Caco2 LS147T MUC1 Expression Restored By Demethylation but not histone re-acetylation Demethylation and histone re-acetylation, but not synergistically Demethylation but not histone re-acetylation Histone re-acetylation, somewhat, perhaps synergy demethylation Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. 2008 American Association for Cancer Research. - 17 -

Both DNA Methylation & Histone Modification Play Roles MUC1 Expression & Regulation in Different Cancer Cell Lines Do MUC1 Methylation Status and Histone Code Correlate with Gene Expression? Cell Line Methylated? Histone Marks PANC1 Y (Inactive Euchromatin) LS147T N (Heterochromatin) MDA-MB-453 Y Heterochromatin Caco2 Y Heterochromatin Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. 2008 American Association for Cancer Research. - 18 -

Model for Epigenetic Regulation of Gene Expression Unmethylated DNA + Acetylated H3 = Strong Expression Methylated DNA + Acetylated H3 = Modest Expression Unmethylated DNA + Methylated H3 = Modest Expression Methylated DNA + Methylated H3 = Very Low Expression Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. 2008 American Association for Cancer Research. - 19 -

SUMMARY.Epigenetic Regulation of Gene Expression Important to Research in Cancer & Toxicology Tissue-Specific Expression & Development Your research field. The Code DNA methylation inhibits expression; DNA demethylation re-activates Histone acetylation permits while histone methylation suppresses expression DNA methylation & histone modification statuses both contribute & synergize.methods & Reagents Available & Well-Established DNA Methylation Analysis Pyrosequencing, Real-Time PCR Reverse methylation with 5-aza-C In vivo DNA-Protein Interactions Analysis Chromatin Immunoprecipitation (histone modifications or TFs) Real-Time PCR Re-acetylate histones with HDAC inhibitor TSA - 20 -

How can you get started analyzing epigenetics? QIAGEN Epigenetic Detection Assay Technologies.DNA Methylation Analysis of Gene or Pathway of Interest Pyrosequencing QIAGEN PyroMark CpG Assays Real-Time PCR QIAGEN EpiTect Methyl II PCR Assays & Arrays.Chromatin Immunoprecipitation (ChIP) for Histone Marks & TF Binding Real-Time PCR QIAGEN EpiTect ChIP PCR Assays & Arrays.Knock Down Expression of Histone Modification Enzymes FlexiTube sirna & FlexiPlate sirna SureSilencing shrna Plasmids.Analyze Expression of Gene or Pathway of Interest RT 2 Profiler PCR Arrays Epigenetic Chromatin Modification Enzymes Epigenetic Chromatin Remodeling Factors Nearly 150 application, disease, pathway-focused arrays RT 2 qpcr Primer Assays & QuantiTect Primer Assays - 21 -

Helping you get started More Technology-Focused Webinars.Profile the Methylation Status of Multiple Genes Simultaneously Without Bisulfite Monday, June 18, 2012 at 5:00 PM Eastern Wednesday, July 25, 2012 at 1:00 PM Eastern.Chromatin Immunoprecipitation and Real-Time PCR Technology Overview Thursday, July 12, 2012 at 1:00 PM Eastern.PCR Arrays: The Real Pioneer in Real-Time PCR Analysis of Biological Pathways Monday, July 2, 2012 at 1:00 PM Eastern.Knock Down Your Favorite Genes or Pathways with Ease and Confidence Tuesday, June 26, 2012 at 9:30 AM Eastern Thursday, July 26, 2012 at 1:00 PM Eastern.RNAi Screening Experiments: Challenges and Solutions Friday, June 29, 2012 at 1:00 PM Eastern Monday, July 9, 2012 at 1:00 PM Eastern - 22 -

Chromatin-Based Regulation of Gene Expression Q&A.George J. Quellhorst, Jr., PhD.Associate Director, R&D.Biological Content Development