Brian P. O’ConnorBrian P. O’ConnorCenter for Genes, Environment & HealthCenter for Genes, Environment & Health

National Jewish HealthNational Jewish Health

Regulation of Cell Identity Regulation of Cell Identity by a Changing Epigenetic Landscapeby a Changing Epigenetic Landscape

Epi- (Epi- (greek: “over, above”) greek: “over, above”) geneticgenetic: : heritable changes in heritable changes in gene expression via mechanisms that are separate from the gene expression via mechanisms that are separate from the DNA sequence and regulated by environmental signals.DNA sequence and regulated by environmental signals.

The processes that controls Chromatin architecture: The processes that controls Chromatin architecture:

Epigenetic regulation of Cellular IdentityEpigenetic regulation of Cellular Identity

Histone ModificationsHistone Modifications

DNA MethylationDNA Methylation CpG islands: Repression of gene transcriptionCpG islands: Repression of gene transcriptionOther areas: More ComplicatedOther areas: More Complicated

Multiple types of modificationsMultiple types of modifications Multiple residues at the N-terminal tailsMultiple residues at the N-terminal tails Combinations of modifications either Combinations of modifications either

support or repress transcriptionsupport or repress transcription

Epi- (Epi- (greek: “over, above”) greek: “over, above”) geneticgenetic: : heritable changes in heritable changes in gene expression via mechanisms that are separate from the gene expression via mechanisms that are separate from the DNA sequence and regulated by environmental signals.DNA sequence and regulated by environmental signals.

Epigenetic regulation of Cellular IdentityEpigenetic regulation of Cellular Identity

Cellular-level:Cellular-level:

EnvironmentEnvironment HeritableHeritable

The tissue microenvironmentThe tissue microenvironment(Hypoxia)(Hypoxia)

Nutrition, Behavior, Pollution,Nutrition, Behavior, Pollution,Sun light, Toxins, Circadian Sun light, Toxins, Circadian rhythms, Viruses, Bacteriarhythms, Viruses, Bacteria

Organism level:Organism level:

Reproduction of Reproduction of epigenetic marks from epigenetic marks from parent cell to daughter parent cell to daughter cell during mitosiscell during mitosis

Parent to child to Parent to child to grandchild?grandchild?

Nucleosomes are the basic building block of Chromatin

Rodenhiser& Mann CMAJ 2006 174(3):341

Luger, K. et al., Nature, Vol.389:251-260. 1997

Post-translation Modifications to the Histone Tails Post-translation Modifications to the Histone Tails

Histone Tails can be Histone Tails can be modified leading to modified leading to changes in chromatin changes in chromatin structure that enhance structure that enhance or inhibit transcriptionor inhibit transcription

Histone Posttranslational Modifications: Defining the Epigenetic Code

HistoneHistonetailtail

Stop

GoAcetylationAcetylation

MethylationMethylation

Histone 3MethylationMethylation

Context matters for the “Code”

The Epigenetic Code: The Epigenetic Code: Combinations of Histone Modifications regulate TranscriptionCombinations of Histone Modifications regulate Transcription

K27meK27me33 K27meK27me33 K27meK27me33

K4meK4me33 K4meK4me33 K4meK4me33

Histone Mark:

Gene Identity:

Repressive

Non-lineage specific gene Lineage specific gene; Enhancer; Regulatory regions

Monovalent, Permissive, ActiveBivalent, Poised

K4meK4me33 K4meK4me33 K4meK4me33

Lineage specific Transcription Factors

Transcription: Off or Low On

K27meK27me33K27meK27me33 K27meK27me33

K4meK4me11K4meK4me33K4meK4me33

The Epigenetic Code regulates Chromatin Structure and Gene Transcription

K27meK27me33 K27meK27me33 K27meK27me33

K4meK4me33

K27meK27me33

K4meK4me33K4meK4me33K4meK4me33

K27meK27me33

K4meK4me33

BivalentBivalent

K4meK4me11

K36meK36me33

K4meK4me33

K27AcK27Ac

K4meK4me33

K27AcK27Ac

The Epigenetic Code regulates Chromatin Structure and Gene Transcription

TF

NFR: Nucleosome Free RegionNFR: Nucleosome Free RegionPol II

Complex

OpenOpen

TF

K4meK4me11

K36meK36me33

K4meK4me33

K27AcK27Ac

K4meK4me33

K27AcK27Ac

The Epigenetic Code regulates Chromatin Structure and Gene Transcription

Transcription Initiation

TF TF Pol IIComplex

ActiveActive

TF

Stem Cell ProgenitorProgenitorCellsCells

Differentiated CellDifferentiated Cell

Epigenetic Regulation of Cell IdentityEpigenetic Regulation of Cell Identity

microenvironment

Bivalent HistoneBivalent HistoneSignatureSignature

Monovalent HistoneMonovalent HistoneSignatureSignature

Malleable IdentityMalleable Identity Fixed IdentityFixed Identity

Nucleosome “replication” during S Phase: Nucleosome “replication” during S Phase: Inheritance of Histone Marks through MitosisInheritance of Histone Marks through Mitosis

RegulatoryProtein

X-ChIP: Chromatin ImmunoPrecipitation AssasyX-ChIP: Chromatin ImmunoPrecipitation Assasy

Assay to examine in vivo DNA-protein interaction dynamics:

Step 1:Step 1: Cross-link the DNA and protein

Step 2:Step 2: Shear the DNA with Sonication

Step 3:Step 3:

Use Specific Antibody to target specific Protein-DNA domains: Regulatory proteins, Transcription Factors, Histone Modifications, etc.

Step 4:Step 4: Use Magnetic Beads to isolate Ab-Protein-DNA fragments

Step 5:Step 5:Elute, Reverse the Cross-links and purify the DNA, which is ready for analysis

Analyzing ChIP isolated DNAAnalyzing ChIP isolated DNA

ABI 7900: qPCR analysis ABI Solid4: Next-Gen Sequencing

Targeted analysis of specific genomic loci

Genome-Wide analysis

ChIP-qPCRChIP-qPCR ChIP-seqChIP-seq

ChIP-Seq analysis: Histones regulate cell differentiationChIP-Seq analysis: Histones regulate cell differentiation

Stem Cell:Non-Expressed

Gene

Neural Progenitor:Expressed

Gene

Goldberg et al., Cell, 140: 678-691. 2010

Bi-Valent No Transcription

Egfr

Mono-ValentActive Transcription

Goldberg et al., Cell, 140: 678-691. 2010

Mapping the Epigenetic Landscape: Cell-type specific histone signatures

CLPCLP

HSC

Bone Marrow Periphery

CMP

Epigenetic regulation of Immune Cell Identity

CCCC

TTFHFH

CCCC

CBCB

CBCB

CBCBCBCB

CBCB

CBCB

TTFHFHTTFHFH

TTFHFH

FDCFDC

Germinal Center

Tissue microenvironment

DCSub-types

Memory

Plasma Cell

Malleable IdentityMalleable Identity Fixed IdentityFixed Identity

Lung

CD103+ CD103+ Lung DCLung DC

Bone Marrow DC Progenitors

CD11b+CD11b+Lung DCLung DC

Epigenetic Regulation of Dendritic Cell DevelopmentEpigenetic Regulation of Dendritic Cell Development

1) 2)2)

3)DC proDC pro

Randolph; Rudensky; Nussenzweig: Science, Vol324, 17April2009.

Edelson… & Murphy, JEM Vol.207, 4:823, 2010.

Ginhoux, … & Merad, JEM Vol.206,13:3115, 2009.

Irf8

Irf4

CD103+ DC CD11b+ DCDC progenitor

ChIP analysis of Irf4 Core Promoter RegionChIP analysis of Irf4 Core Promoter Region

Irf4K4meK4me33Irf4K27meK27me33

CD103+ DC Irf4 promoter is repressive

CD11b+ DC Irf4 promoter is active

BM DC progenitor Irf4 promoter is bi-valent/ poisedIrf8

Irf4

CD103+ DC CD11b+ DCDC progenitor

DC proDC pro

CD103+ DC

CD11b+ DC

ChIP Analysis of Irf4 Promoter:

Irf4 Gene Expression Changes during DC development

DC proDC pro

CD103+ DC CD11b+ DCDC progenitor

Bi-valent Repressed Active

Irf4K27meK27me33

K4meK4me33

-500bp NFR

KDM H3K4KMT inactivity

DC proDC pro

CD103+ DC

CD11b+ DC

DC proDC pro

CD103+ DC

CD11b+ DC

Irf4 Locus Irf8 Locus

Summary: Unique Histone Signatures Characterize Specific Cell Lineages

KMT H3K4KMT H3K4

KDM H3K27KMT inactivity

KDM H3K4KMT decreased activity

Hypoxia controls Histone Modifications at Oxygen-Regulated GenesHypoxia controls Histone Modifications at Oxygen-Regulated Genes

Direct Model In-Direct Model

Wenger RH, Journal of Exp Bio; 203:1253-1263, 2000.

Krieg et al., Mol and Cell Bio; 30,1:344-353, 2010.

What have we learned ?

The cell differentiation process can utilize opposing types of histone modifying enzymes.

The local chromatin structure of genes are unique to each cell lineage.

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.

Post-translational histone modifications alter chromatin structure and create “landing pads” for Chromatin binding proteins/DNA-binding proteins/transcription factor binding at specific loci throughout the genome. Coding & Non-Coding.