Embed Size (px)
Citation preview
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 2
Sample & Assay Technologies Chromatin = DNA + DNA Binding Proteins
Structural (Histones) & Functional (Transcription F actors)
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 3
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 4
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 5
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 6
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 7
Sample & Assay Technologies The Histone Code
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 8
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 9
Sample & Assay Technologies DNA Methylation & Histone Modification Interplay
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 10
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 11
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 12
Sample & Assay Technologies Promoter Methylation Decreases Gene Expression
ERBB4 (HER4) in Breast Cancer Cell Lines
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 13
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 14
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 15
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 16
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 17
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 18
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 19
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 20
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 21
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 22
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
More Resources to Help You Technical Support Email: [email protected] Webinar Related Questions Email: [email protected]
Also, please remember to complete our survey after the webinar.
With Your First or Next Order of Reagents
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 23
Sample & Assay Technologies
Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD Associate Director, R&D, Biological Research Content Development
Q&A
