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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.

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Brian P. O’Connor Center for Genes, Environment & Health National Jewish Health

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

  2. 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  Other areas: More Complicated DNA Methylation • Multiple types of modifications • Multiple residues at the N-terminal tails • Combinations of modifications either support or repress transcription Histone Modifications

  3. 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. Heritable Environment Nutrition, Behavior, Pollution, Sun light, Toxins, Circadian rhythms, Viruses, Bacteria Organism level: Parent to child to grandchild? Reproduction of epigenetic marks from parent cell to daughter cell during mitosis Cellular-level: The tissue microenvironment (Hypoxia)

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

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

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

  7. 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 Lineage specific gene; Enhancer; Regulatory regions Non-lineage specific gene Gene Identity: Lineage specific Transcription Factors

  8. The Epigenetic Code regulates Chromatin Structure and Gene Transcription Bivalent K4me1 K4me3 K4me3 K4me3 K4me3 K27me3 K4me3 K27me3 K4me3 K4me3 K27me3 K27me3 K27me3 K27me3 K27me3 K27me3

  9. The Epigenetic Code regulates Chromatin Structure and Gene Transcription K36me3 Open K27Ac K27Ac K4me3 K4me1 K4me3 NFR: Nucleosome Free Region Pol II Complex TF TF

  10. The Epigenetic Code regulates Chromatin Structure and Gene Transcription K36me3 K27Ac K27Ac Active K4me3 K4me1 K4me3 Transcription Initiation Pol II Complex TF TF TF

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

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

  13. 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 Use Specific Antibody to target specific Protein-DNA domains: Regulatory proteins, Transcription Factors, Histone Modifications, etc. Regulatory Protein Step 3: Use Magnetic Beads to isolate Ab-Protein-DNA fragments Step 4: Elute, Reverse the Cross-links and purify the DNA, which is ready for analysis Step 5:

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

  15. ChIP-Seq analysis: Histones regulate cell differentiation Egfr Stem Cell: Non-Expressed Gene Bi-Valent No Transcription Neural Progenitor: Expressed Gene Mono-Valent Active Transcription Goldberg et al., Cell, 140: 678-691. 2010

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

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

  18. Epigenetic Regulation of Dendritic Cell Development DC pro Irf8 Bone Marrow DC Progenitors 1) CD103+ Lung DC 2) Lung CD11b+ Lung DC 3) Irf4 Ginhoux, … & Merad, JEM Vol.206,13:3115, 2009. Edelson… & Murphy, JEM Vol.207, 4:823, 2010. Randolph; Rudensky; Nussenzweig: Science, Vol324, 17April2009.

  19. ChIP analysis of Irf4 Core Promoter Region Irf4 Irf4 DC pro DC progenitor CD103+ DC CD11b+ DC DC progenitor CD103+ DC CD11b+ DC K4me3 K27me3 BM DC progenitor Irf4 promoter is bi-valent/ poised CD103+ DC Irf8 CD103+ DC Irf4 promoter is repressive CD11b+ DC Irf4 CD11b+ DC Irf4 promoter is active

  20. Irf4 Gene Expression Changes during DC development DC pro DC progenitor CD103+ DC CD11b+ DC K4me3 ChIP Analysis of Irf4 Promoter: Bi-valent Repressed Active Irf4 K27me3 NFR -500bp

  21. Summary: Unique Histone Signatures Characterize Specific Cell Lineages Irf4 Locus Irf8 Locus KDM H3K27 KMT inactivity KDM H3K4 KMT inactivity DC pro DC pro CD103+ DC CD103+ DC CD11b+ DC CD11b+ DC KDM H3K4 KMT decreased activity KMT H3K4

  22. Hypoxia controls Histone Modifications at Oxygen-Regulated Genes In-Direct Model Direct Model Krieg et al., Mol and Cell Bio; 30,1:344-353, 2010. Wenger RH, Journal of Exp Bio; 203:1253-1263, 2000.

  23. What have we learned ?  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.  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.

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