Reprogramming of the Genome by Toxic Injury Ken Ramos, MD, PhD, ATS

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2 Reprogramming of the Genome by Toxic Injury Ken Ramos, MD, PhD, ATS Professor of dicine, University of Arizona Health Sciences Center & Associate Vice President for Precision Health Sciences Office of the Senior Vice President for Health Sciences

3 The Human Genome The human genome is stored on 23 chromosome pairs and encompasses over 3 billion DNA base pairs. Protein coding sequences comprise less than 2% of the genome. Aside from genes and regulatory sequences, the genome contains vast regions of DNA with poorly characterized functions. Coding regions (~2%) Repetitive DNA e.g. DNA Transposons & Retrotransposons (~44%)

4 LINE-1 Retroelements 5 UTR: Contains a promoter region with regulatory sequences that bind YY1, RunX3, and E2F/Rb ORF1: RNA-binding protein that shuttles L1 RNA ORF2: Endonuclease and reverse transcriptase activities ~600,000 copies per genome (17%) ~100 full length active in the human genome Silenced by DNA methylation and reactivated by reversal of epigenetic marks.

5 5 Executive Life Cycle Committee of LINE-1 Toxic injury by BaP reactivates L1 retrotransposition via epigenetic mechanisms

6 Genotoxicity, Oxidative Stress AHR, Epigenetics

7 What is Epigenetics? Heritable changes in gene expression without changes in DNA sequence. Assembly of protein complexes on chromatin to activate or silence genes. If not properly controlled can mediate adverse health effects. Environmental toxicants can alter epigenome to cause disease. DNA thylation and Histone Modifications are two well characterized epigenetic pathways.

8 L1 Silencing During Embryonic Development 5 UTR ORF-1 ORF-2 DNMTs 5 UTR ORF-1 ORF-2 Sequence-specific TF RNA Pol complex Activator Unmethylated CpG Sequence-specific repressor thyl-binding protein DNA methyl-transferase thylated CpG 5 -CpG-3 3 -GpC-5 Active DNMTs CH3 5 -CpG-3 3 -GpC-5 Inactive

9 L1 Silencing by E2F/RB 5 UTR ORF-1 ORF-2 Rb is recruited to both human and mouse L1 5 UTR in vivo. E2F1 and E2F4 bind to both human and mouse L1 5 UTR in vivo E2F binding to L1 is Rb-independent. Rb prevents cells from replicating damaged DNA by inhibiting progression from G1 to S. Rb recruits HDACs that inhibit E2F and facilitate condensation of nucleosomes into heterochromatin. Mutation Research, 2009

10 L1 Retroelement Silencing: RB Knockdown 5 UTR ORF- 1 ORF- 2 HDACs HMTases (Suv) 5 UTR ORF- 1 ORF- 2 ü Rb loss leads to overexpression of endogenous L1 in primary cells. ü Pharmacological inhibilon of HDACs increases L1 expression in human and wild type MEFs, but not Rb- null cells.

11 Executive Committee Integration of the Evidence Nucleosomal Histone Modifications (thylation/demethylation and Acetylation/Deacetylation) HDACs HMTs E2F/RB/AHR CP2 MBDs L1 Silencing and Reactivation DNA thylation (Cytosine thylation and Hydroxymethylation) CP2 H3K9 3 H4K20 3 H4K20 3 ORF1 MBDs H3K9 3 ATG DNMT1 DNMT3a/b CP2 De Novo DNMTs Maintenance DNMT HDACs DNMT1 Transcription Factor Recruitment to L1 DNA Binding Sites H4K20 3 H4K20 3 H3K9 3 H3K9 3 H4K20 3 H4K20 3 H3K9 3 H3K9 3 H4K20 3 H4K20 3 DNMT3a/b DNMT1 Ramos et al., 2013

12 Impacts of L1 in the Human Genome Evolution Insertion, Duplication, Inversion Mutations Inhibition of Gene Transcription Developmental Programming Alternative Splicing Sites Exon Skipping Alternative Promoters DNA Strand Breaks DNA Repair Genetic Reprogramming

13 Transition from Normal to Disease Phenotypes : A Precision dicine Perspective L1? Deficits in cellular differentiation and proliferation Recapitulation of developmental programming

14 Overexpression of L1 in Epithelial Cells Mimics the Actions of BaP L1 Associated with EMT in HepG2 Cells and Bronchial Epithelial Cells Deficits are associated with disruption of L1 genetic regulatory network. Molecular Oncology, 2014

15 L1 Regulatory Networks Chi3l3 Clic3 M12039 ELD Prot Ty RiKEN Icam1 Gnal2 MGST1 VCA RIKEN cdna Pkia TEK EST Syn BP Slc34a2 Rgs2 NMO Cyp2a4 RNA Pol II Agl Evi 2a Rnpc2 Col3a1 Cdo1 Vamp3 Dbp Dnajb9 Sgpl1 Myl7 Pah Gja1 Cxcl1 Ccl2

16 Biological Inference Genetic knockdown of AHR or other targets by si- RNA. Gene expression within the predicted regulatory network measured by RT- PCR PAS proteins identified as members of the L1regulatory network

17 L1 and Precision dicine 5 UTR ORF 1 ORF 2 3 UTR Poly-A Antisense Promoter 1K 2K 3K 4K 5K 6K PM is a medical model that emphasizes use of genetic and molecular data to optimize prevention and treatment. Current standards of care are based on epidemiological data and not genetic variability or molecular taxonomy of disease. Molecular assays can be used to stratify disease, select proper medication, or tailoring dosages to prevent adverse outcomes and toxicity. L1 is highly polymorphic, with varying levels of L1 activity in response to toxic injury correlated to disease onset or severity in human populations.

18 Closing Remarks The Future of Toxicology Acknowledgements