chromatin regulation 2013
TRANSCRIPT
Sample & Assay Technologies
Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD Associate Director, R&D, Biological Research Content Development
Sample & Assay Technologies 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
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Sample & Assay Technologies Chromatin = DNA + DNA Binding Proteins
Structural (Histones) & Functional (Transcription F actors)
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Matouk, C. C. et al. Circ Res 2008;102:873-887. Copyright © 2008 American Heart Association
Sample & Assay Technologies Transcriptional Regulation of Gene Expression
An Evolving Picture
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Structural Gene
Proximal PromoterDistal Promoter
TSS (+1)
RNAPol II
SWI/SNF orINO80 orMediatorComplex
+
–
TBP
p53 BS NFκB BS
TF Activating Signal Transduction Cascades
p53
NFκB
OAc OAc OAcMeMe MeHistones
THE Gene Expression Regulation Frontier!Highly Dynamic Promoter Context
?
DNA MethylationHistone Modification
Me MeMe Me
mRNA or miRNA
Sample & Assay Technologies Writing, Reading, Erasing DNA Methylation
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Cytosine
N
N
NH2
ON
N
NH2
O
R
H
S -Enz
H
CH3
N
N
NH2
O CH3
+ DNMT - DNMT
AdoMet AdoHcy
5-Methyl-Cytosine
Herman JG, Baylin SB. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 20:2042. © 2003 Massachusetts Medical Society
1111 2222 3333
1111 2222 3333
Normal
Cancer
Promoter Region
Expression
No Expression
Modification of cytosine in CpG dinucleotide at 5-position with methyl (CH3) groupWritten by DNA Methyltransferases
DNMT1: Maintenance MethylationDNMT3A & DNMT3B: De Novo Methylation
Read byTranscription FactorsMethyl DNA Binding ProteinHistone Modification Enzymes
Erased by … DNA Demethylases?
Tumor Suppressor Genes
Sample & Assay Technologies DNA Methylation Dependent Transcriptional Repression
Possible Molecular Mechanisms
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Singal R and Ginder GD (1999) DNA Methylation. Blood 93:4059. © 1999 by The American Society of Hematology
Sample & Assay Technologies 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
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Sample & Assay Technologies The Histone Code
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AcetylationH3ac Active EuchromatinH3K9ac Active EuchromatinH4ac 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
Sample & Assay Technologies Writing, Reading, 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
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Sample & Assay Technologies DNA Methylation & Histone Modification Interplay
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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
Sample & Assay Technologies DNA Methylation & Histone Modification Interplay
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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
Sample & Assay Technologies Examples of Experimental Studies
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
Questions Asked & Methods Used
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Sample & Assay Technologies Promoter Methylation Decreases Gene Expression
ERBB4 (HER4) in Breast Cancer Cell Lines
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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
RT-PCR: HER4
Bisulfite Pyrosequencing Results
More MethylationLess Expression
Why are some breast cancers HER4 positive and some HER4 negative?
Sample & Assay Technologies Methylation Inhibits TF Binding but Recruits MeCP2
CREB, ATF2 and MeCP2 Binding to Insulin Promoter
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Kuroda A et al. (2009) Insulin gene expression is regulated by DNA methylation. PLoS One. 4:e6953. © 2009
IP: Various TFsPCR: Endogenous Promoter
IP: Various TFsPCR: Transfected Reporter Plasmids
Chromatin Immunoprecipitation followed by PCRfrom NIT-1 mouse insulinoma cell line
What are the consequences of tissue-specific methylation of the insulin promoter?
Sample & Assay Technologies Is Increased Gene Expression & TF Binding Epigenetic?
Ethanol & 5-aza-C Similarly Increase GRIN2B Express ion & TF Binding
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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
CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawalCIEW5: CIE + 5-day withdrawal5’AZA: 5-Azacytidine
RT-PCR: GRIN2B
Chromatin IummunoprecipitationIP: Various TFsPCR: GRIN2B Promoter TFBS
Primary cortical cultured neurons
Why does ethanol activate expression of GRIN2B?
Sample & Assay Technologies Removing Methylation Restores Gene Expression
Ethanol Treatment Decreases GRIN2B Methylation, DNM T1 Expression
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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
Bisulfite Pyrosequencing Results
RT-PCR: DNMT1
SAM = S-adenosyl-L-methionine, DNMT substrate– Rescues hypomethylation
Why does ethanol activate expression of GRIN2B?
CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawalCIEW5: CIE + 5-day withdrawal5’AZA: 5-Azacytidine
Sample & Assay Technologies Epigenetic Regulation Keeps Oncogenes Suppressed
MUC1 Expression & Regulation in Different Cancer Ce ll Lines
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Cell Line MUC1 Expression Restored By …PANC1 Demethylation but not histone re-acetylationMDA-MB-453 Demethylation and histone re-acetylation, but not synergisticallyCaco2 Demethylation but not histone re-acetylationLS147T Histone re-acetylation, somewhat, perhaps synergy demethylation
Why do some cancers NOT express MUC1, a potential marker for malignancy?
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.
Sample & Assay Technologies Both DNA Methylation & Histone Modification Play Roles
MUC1 Expression & Regulation in Different Cancer Ce ll Lines
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Cell Line Methylated? Histone MarksPANC1 Y (Inactive Euchromatin)LS147T N (Heterochromatin)MDA-MB-453 Y HeterochromatinCaco2 Y Heterochromatin
Do MUC1 Methylation Status and Histone Code Correlate with Gene 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.
Sample & Assay Technologies Model for Epigenetic Regulation of Gene Expression
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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.
Sample & Assay Technologies 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
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Sample & Assay Technologies How can you get started analyzing epigenetics?
DNA Methylation Analysis of Gene or Pathway of Inte rest� Pyrosequencing
� QIAGEN PyroMark CpG Assays� Real-Time PCR
� QIAGEN EpiTect Methyl II PCR Assays & Arrays Chromatin Immunoprecipitation (ChIP) for Histone Ma rks & TF Binding� Real-Time PCR
� QIAGEN EpiTect ChIP PCR Assays & Arrays Knock Down Expression of Histone Modification Enzym es� FlexiTube siRNA & FlexiPlate siRNA� SureSilencing shRNA Plasmids Analyze Expression of Gene or Pathway of Interest� RT2 Profiler PCR Arrays
� Epigenetic Chromatin Modification Enzymes� Epigenetic Chromatin Remodeling Factors� Nearly 150 application, disease, pathway-focused arrays
� RT2 qPCR Primer Assays & QuantiTect Primer Assays
QIAGEN Epigenetic Assay Technologies
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Sample & Assay Technologies Helping you get started …
Profile the Methylation Status of Multiple Genes Simultaneously Without Bisulfite� Tuesday, April 23, 2013 at 9:30 AM Eastern US
Chromatin Immunoprecipitation and Real-Time PCR Technology Overview� Wednesday, May 8, 2013 at 1:00 PM Eastern US
PCR Arrays: The Real Pioneer in Real-Time PCR Analysis of Biological Pathways� Monday, April 22, 2013 at 9:30 AM Eastern US
Knock Down Your Favorite Genes or Pathways with Ease and Confidence� Friday, April 19, 2013 at 1:00 PM Eastern US
More Technology-Focused Webinars
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Sample & Assay Technologies Helping you get started …
Get 20% any of the following products:� EpiTect Methyl II PCR Assays & Arrays� EpiTect ChIP PCR Assays & Arrays� SureSilencing shRNA Plasmids� RT2 Profiler PCR Arrays� RT2 qPCR Primer Assays
PLUS Auto Emergency Kit! Simply refer to PROMO CODE: FDK-WN20W22
Only available in US & Canada; Expires April 30, 2013
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Sample & Assay Technologies
Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD Associate Director, R&D, Biological Research Content Development
Q&A