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

2. 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: Epigenetic regulation of Cellular Identity Histone Modifications DNA Methylation  CpG islands: Repression of gene transcription  Other areas: More Complicated  Multiple types of modifications  Multiple residues at the N-terminal tails  Combinations of modifications either support or repress transcription

3. Epi- (greek: “over, above”) genetic: heritable changes in gene expression via mechanisms that are separate from the DNA sequence and regulated by environmental signals. Epigenetic regulation of Cellular Identity Cellular-level: Environment Heritable The tissue microenvironment (Hypoxia) Nutrition, Behavior, Pollution, Sun light, Toxins, Circadian rhythms, Viruses, Bacteria Organism level: Reproduction of epigenetic marks from parent cell to daughter cell during mitosis Parent to child to grandchild?

4. Nucleosomes are the basic building block of Chromatin Rodenhiser& Mann CMAJ 2006 174(3):341

5. Luger, K. et al., Nature, Vol.389:251-260. 1997 Post-translation Modifications to the Histone Tails

6. Histone Tails can be modified leading to changes in chromatin structure that enhance or inhibit transcription Histone Posttranslational Modifications: Defining the Epigenetic Code Histonetail Stop Go Acetylation Methylation Histone 3 Methylation Context matters for the “Code”

7. The Epigenetic Code: Combinations of Histone Modifications regulate Transcription K27me 3 K4me 3 Histone Mark: Gene Identity: Repressive Non-lineage specific gene Lineage specific gene; Enhancer; Regulatory regions Monovalent, Permissive, ActiveBivalent, Poised K4me 3 Lineage specific Transcription Factors Transcription:Off or LowOn

8. K27me 3 K4me 1 K4me 3 The Epigenetic Code regulates Chromatin Structure and Gene Transcription K27me 3 K4me 3 K27me 3 K4me 3 K27me 3 K4me 3 Bivalent

9. K4me 1 K36me 3 K4me 3 K27Ac K27Ac The Epigenetic Code regulates Chromatin Structure and Gene Transcription TF NFR: Nucleosome Free Region Pol II Complex Open TF

10. K4me 1 K36me 3 K4me 3 K27Ac K27Ac The Epigenetic Code regulates Chromatin Structure and Gene Transcription Transcription Initiation TF Pol II Complex Active TF

11. Stem Cell ProgenitorCells Differentiated Cell Epigenetic Regulation of Cell Identity microenvironment Bivalent Histone Signature Monovalent Histone Signature Malleable Identity Fixed Identity

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

13. Regulatory Protein 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

14. Analyzing ChIP isolated DNA ABI 7900: qPCR analysisABI Solid4: Next-Gen Sequencing Targeted analysis of specific genomic loci Genome-Wide analysis ChIP-qPCRChIP-seq

15. ChIP-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-Valent Active Transcription

16. Goldberg et al., Cell, 140: 678-691. 2010 Mapping the Epigenetic Landscape: Cell-type specific histone signatures

17. CLP HSC Bone MarrowPeriphery CMP Epigenetic regulation of Immune Cell Identity CC T FH CC CB CB CB CB CB CB FDC Germinal Center Tissue microenvironment DC Sub-types Memory Plasma Cell Malleable Identity Fixed Identity

18. Lung CD103+ Lung DC Bone Marrow DC Progenitors CD11b+ Lung DC Epigenetic Regulation of Dendritic Cell Development 1) 2) 3) DC 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

19. CD103+ DCCD11b+ DCDC progenitor ChIP analysis of Irf4 Core Promoter Region Irf4 K4me 3 Irf4 K27me 3 CD103+ DC Irf4 promoter is repressive CD11b+ DC Irf4 promoter is active BM DC progenitor Irf4 promoter is bi-valent/ poised Irf8 Irf4 CD103+ DCCD11b+ DCDC progenitor DC pro CD103+ DC CD11b+ DC

20. ChIP Analysis of Irf4 Promoter: Irf4 Gene Expression Changes during DC development DC pro CD103+ DCCD11b+ DCDC progenitor Bi-valentRepressedActive Irf4 K27me 3 K4me 3 -500bp NFR

21. KDM H3K4 KMT inactivity DC pro CD103+ DC CD11b+ DC DC pro CD103+ DC CD11b+ DC Irf4 LocusIrf8 Locus Summary: Unique Histone Signatures Characterize Specific Cell Lineages KMT H3K4 KDM H3K27 KMT inactivity KDM H3K4 KMT decreased activity

22. Hypoxia 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.

23. 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.