Transcript Regulation of Cell Identity by a Changing Epigenetic Landscape Brian P. O’Connor Center for Genes, Environment & Health National Jewish Health.

Regulation of Cell Identity
by a Changing Epigenetic Landscape
Brian P. O’Connor
Center for Genes, Environment & Health
National Jewish Health
Epigenetic regulation of Cellular Identity
Epi- (greek: “over, above”) genetic: heritable changes in
gene expression via mechanisms that are separate from the
DNA sequence and regulated by environmental signals.
The processes that controls Chromatin architecture:
 CpG islands: Repression of gene transcription
DNA Methylation  Other areas: More Complicated
Histone Modifications
 Multiple types of modifications
 Multiple residues at the N-terminal tails
 Combinations of modifications either
support or repress transcription
Epigenetic regulation of Cellular Identity
Epi- (greek: “over, above”) genetic: heritable changes in
gene expression via mechanisms that are separate from the
DNA sequence and regulated by environmental signals.
Environment
Organism level: Nutrition, Behavior, Pollution,
Sun light, Toxins, Circadian
rhythms, Viruses, Bacteria
Cellular-level:
The tissue microenvironment
(Hypoxia)
Heritable
Parent to child to
grandchild?
Reproduction of
epigenetic marks from
parent cell to daughter
cell during mitosis
Nucleosomes are the basic building block of Chromatin
Rodenhiser& Mann CMAJ 2006 174(3):341
Post-translation Modifications to the Histone Tails
Luger, K. et al., Nature, Vol.389:251-260. 1997
Histone Posttranslational Modifications:
Defining the Epigenetic Code
Context matters for the “Code”
Histone 3
Histone
tail
Histone Tails can be
modified leading to
changes in chromatin
structure that enhance
or inhibit transcription
Go
Stop
Acetylation
Methylation
Methylation
The Epigenetic Code:
Combinations of Histone Modifications regulate Transcription
K4me3
K4me3
K4me3
K27me3
K27me3
K27me3
K4me3
K4me3
K4me3
Histone Mark:
Repressive Bivalent, Poised
Monovalent, Permissive, Active
Transcription:
Off or Low
On
Gene Identity: Non-lineage specific gene
Lineage specific Transcription Factors
Lineage specific gene; Enhancer;
Regulatory regions
The Epigenetic Code regulates Chromatin Structure and Gene Transcription
Bivalent
K4me3
K27me3
K4me3
K27me3
K4me3
K27me3
K4me3
K27me3
K4me3
K27me3
K4me3
K27me3
K4me3
K27me3
K4me1
K27me3
The Epigenetic Code regulates Chromatin Structure and Gene Transcription
Open
K4me3
K4me3
K4me1
K36me3
K27Ac
K27Ac
TF
NFR: Nucleosome Free Region
Pol II
Complex
TF
The Epigenetic Code regulates Chromatin Structure and Gene Transcription
Active
K4me3
K4me3
K4me1
K36me3
K27Ac
K27Ac
TF
TF TF
Pol II
Complex
Transcription Initiation
Epigenetic Regulation of Cell Identity
Stem Cell
Progenitor
Cells
Differentiated Cell
microenvironment
Bivalent Histone
Signature
Monovalent Histone
Signature
Malleable Identity
Fixed Identity
Nucleosome “replication” during S Phase:
Inheritance of Histone Marks through Mitosis
X-ChIP: Chromatin ImmunoPrecipitation Assasy
Assay to examine in vivo DNA-protein interaction dynamics:
Step 1:
Cross-link the DNA and protein
Step 2:
Shear the DNA with Sonication
Step 3:
Use Specific Antibody to target
specific Protein-DNA domains:
Regulatory proteins, Transcription
Factors, Histone Modifications, etc.
Step 4:
Use Magnetic Beads to isolate
Ab-Protein-DNA fragments
Step 5:
Elute, Reverse the Cross-links
and purify the DNA, which is
ready for analysis
Regulatory
Protein
Analyzing ChIP isolated DNA
ChIP-qPCR
ABI 7900: qPCR analysis
Targeted analysis of
specific genomic loci
ChIP-seq
ABI Solid4: Next-Gen Sequencing
Genome-Wide analysis
ChIP-Seq analysis: Histones regulate cell differentiation
Egfr
Stem Cell:
Non-Expressed
Gene
Neural Progenitor:
Expressed
Gene
Bi-Valent
No Transcription
Mono-Valent
Active Transcription
Goldberg et al., Cell, 140: 678-691. 2010
Mapping the Epigenetic Landscape:
Cell-type specific histone signatures
Goldberg et al., Cell, 140: 678-691. 2010
Epigenetic regulation of Immune Cell Identity
Bone Marrow
Periphery
DC
Sub-types
CMP
HSC
Tissue
microenvironment
Fixed Identity
Malleable Identity
Germinal Center
Memory
TFH
TFH
CLP
FDC
TFH
TFH
CB
CC
CC
CB CB
CB
CB
CB
Plasma
Cell
Epigenetic Regulation of Dendritic Cell Development
1)
Bone Marrow
DC Progenitors
Irf8
2) CD103+
Lung DC
Lung
DC pro
Irf4
Ginhoux, … & Merad, JEM Vol.206,13:3115, 2009.
Edelson… & Murphy, JEM Vol.207, 4:823, 2010.
Randolph; Rudensky; Nussenzweig: Science, Vol324, 17April2009.
3) CD11b+
Lung DC
ChIP analysis of Irf4 Core Promoter Region
K4me3
Irf4
K27me3
1
Association Normalized to Input
Association Normalized to Input
1
Irf4
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
DC pro
CD103 DCDC
DCBM
progenitor
CD103+
Irf8
CD11b DC
CD11b+
CD103+ DC
DCBM
progenitor
DC pro
CD103+
CD103 DC
CD11b+
CD11b DCDC
BM DC progenitor Irf4 promoter is bi-valent/ poised
CD103+ DC Irf4 promoter is repressive
DC pro
Irf4
CD11b+ DC
CD11b+ DC Irf4 promoter is active
Irf4 Gene Expression Changes during DC development
DC pro
Irf4 mRNA expression
700
600
500
400
300
200
100
0
ChIP Analysis
of Irf4 Promoter:
K4me3
Irf4
K27me3
-500bp
NFR
BM Pro
DC progenitor
CD103+ DCDC
CD103+
Bi-valent
Repressed
CD11b+ DC
CD11b+
DC
Active
Summary: Unique Histone Signatures Characterize
Specific Cell Lineages
Irf4 Locus
Irf8 Locus
KDM H3K27
KMT inactivity
KDM H3K4
KMT inactivity
CD103+ DC
DC pro
CD103+ DC
DC pro
CD11b+ DC
KMT H3K4
CD11b+ DC
KDM H3K4
KMT decreased activity
Hypoxia controls Histone Modifications at Oxygen-Regulated Genes
Direct Model
In-Direct Model
Krieg et al., Mol and Cell Bio; 30,1:344-353, 2010.
Wenger RH, Journal of Exp Bio; 203:1253-1263, 2000.
What have we learned ?
Post-translational histone modifications alter chromatin structure
and create “landing pads” for Chromatin binding proteins/DNAbinding proteins/transcription factor binding at specific loci
throughout the genome. Coding & Non-Coding.
 The local chromatin structure of genes are unique to each cell
lineage.
 The cell differentiation process can utilize opposing types of
histone modifying enzymes.
 The epigenetic regulation of differentiation is affected by local
microenvironments.
 The tissue microenvironment (Inflammation, Hypoxia) can affect
histone modifying enzyme activity, and thus regulates the malleable
process of progenitor cell differentiation to terminally differentiated
cells.