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

2. 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
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3. Sample & Assay Technologies
Chromatin = DNA + DNA Binding Proteins
Structural (Histones) & Functional (Transcription Factors)
Matouk, C. C. et al. Circ Res 2008;102:873-887. Copyright © 2008 American Heart Association
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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4. Sample & Assay Technologies
Transcriptional Regulation of Gene Expression
An Evolving Picture
TF Activating Signal
Transduction Cascades
THE Gene Expression Regulation Frontier!
Highly Dynamic Promoter Context
NFκB
+
p53
?
–
SWI/SNF or
INO80 or
Mediator
Complex
RNA
Pol II
Structural Gene
TSS (+1)
mRNA or miRNA
TBP
p53 BS
NFκB BS
Distal Promoter
Histones
Me
Me
Me
Me Me Me
Me
OAc
OAc
OAc
Proximal Promoter
Histone Modification
DNA Methylation
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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5. Writing, Reading, Erasing DNA Methylation
Sample & Assay Technologies
NH 2
N
N
+ DNMT
O
O
AdoHcy
NH 2
N
- DNMT
CH3
O
C H3
N S -E n z
N
AdoMet
H NH 2
H
N
R
Cytosine
5-Methyl-Cytosine
Modification of cytosine in CpG dinucleotide at 5-position with methyl (CH3) 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?
1
Normal
2
3
Expression
1
2
3
No Expression
Promoter
Region
Tumor Suppressor Genes
Cancer
Herman JG, Baylin SB. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 20:2042.
© 2003 Massachusetts Medical Society
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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6. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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7. 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
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8. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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9. 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
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10. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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11. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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12. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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13. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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14. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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15. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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16. Sample & Assay Technologies
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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17. Sample & Assay Technologies
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.
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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18. Sample & Assay Technologies
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
PANC1
LS147T
MDA-MB-453
Caco2
Methylated?
Y
N
Y
Y
Histone Marks
(Inactive Euchromatin)
(Heterochromatin)
Heterochromatin
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.
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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19. Sample & Assay Technologies
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.
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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20. 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
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21. Sample & Assay Technologies
How can you get started analyzing epigenetics?
QIAGEN Epigenetic 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
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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22. Sample & Assay Technologies
Helping you get started …
More Technology-Focused Webinars
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
Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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23. Sample & Assay Technologies
Helping you get started …
With Your First or Next Order of Reagents
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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Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013
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24. Sample & Assay Technologies
Q&A
Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD
Associate Director, R&D, Biological Research Content Development