R. Piazza (MD, PhD), Dept. of Medicine and Surgery, University of Milano-Bicocca EPIGENETICS

Transcription

1 R. Piazza (MD, PhD), Dept. of Medicine and Surgery, University of Milano-Bicocca EPIGENETICS

2 EPIGENETICS THE STUDY OF CHANGES IN GENE EXPRESSION THAT ARE POTENTIALLY HERITABLE AND THAT DO NOT ENTAIL A CHANGE IN DNA SEQUENCE

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11 EPIGENETIC GENE SILENCING EUCHROMATIN GENOMIC DNA METHYLATION RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY CHROMATIN CONDENSATION (HETEROCHROMATIN)

12 DNA METHYLATION DNA METHYLATION OCCURS AT POSITION 5 (ALMOST) ALWAYS IN A CYTOSINE FOLLOWED BY A GUANINE (CpG DINUCLEOTIDES)

13 PALINDROMIC SEQUENCE! CpG?? m CA GT???? +

14 DNA methylation 3 human DNA methyltransferases DNMT1 DNMT3A DNMT3B maintenance methyltransferases de novo methyltransferases highly expressed at embryo implantation when waves of de novo methylation are occurring in the genome daughter strand daughter strand

15 EUCHROMATIN GENOMIC DNA METHYLATION RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY CHROMATIN CONDENSATION (HETEROCHROMATIN)

16 What is the distribution of CpG sites in a gene? Which methylation pattern is typically found in actively transcribed and in non-expressed genes? Unmethylated CpG site Methylated CpG site

17 CpG islands CpG islands are typically 300-3,000 base pairs in length. They are in and near approximately 40% of promoters of mammalian genes. The CpG island is a region of at least 200 bp, with a G + C percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 60%. Theoretical probability of getting a specific dinucleotide? (e.g. CG o AT)? 1/16 = 6.25% Observed/expected CpG ratio greater than 60% refers to the theoretical probability (6.25%): 60% di 6.25% = ~3.75% Owing to the phenomenon known as m CpG suppression the percentage of CG dinucleotides in the human genome is much smaller: 1% I.e. approximately 3-4-fold greater than the mean percentage of CG dinucleotides in the whole human genome (1%).

18 m CG SUPPRESSION Methyl-Cytosine Thymine OXIDATIVE DEAMINATION?? Unmethylated CpG site Methylated CpG site

19 GENOMIC DNA METHYLATION GENE SILENCING EUCHROMATIN GENOMIC DNA METHYLATION RECRUITMENT OF METHYL-CpG BINDING PROTEINS RECRUITMENT OF COREPRESSOR COMPLEXES WITH DEACETYLASE ACTIVITY CHROMATIN CONDENSATION (HETEROCHROMATIN)

20 Sin3 - NuRD

21 HDACs

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23 Histone Methyl-Transferase (HMT)

24 Methylation of K9 of H3 = gene silencing

25 H4 H3 Mechanism: etylation of H3 or H4 leads to unfolding and accessibility of chromatin (histone acetyltransferases) Methylation of K4 of H3 = active gene expression Methylation of K9 of H3 = gene silencing

26 Mechanism: 1) etylation of H3 or H4 leads to unfolding and accessibility of chromatin (histone acetyltransferases) 2) Methylation of K4 of H3 = active gene expression 3) Methylation of K9 of H3 = gene silencing

27 EPIGENETICS AND CANCER R. Piazza Dept. of Medicine and Surgery University of Milano-Bicocca

28 Historically, Cancer Was Considered to be Driven Mostly by Genetic Changes GENETIC Example: Replication errors Altered DNA sequence X X Altered DNA/mRNA/proteins Oncogenesis Tumor

29 DNA POLYMERASE

30 BOOK: 300 PAGES 1500 CHARs PER PAGE CHARs DNA POLIMERASI HUMAN gdna: CHARs (BASES) SAME AS BOOKS!! CELLS TURN-OVER: BILLION CELLS EACH DAY. HUMAN DNA POLYMERASES MUST READ AND COPY ON AVERAGE 600 BILLION BILLION BASES EACH DAY: I.E. MORE THAN A MILLION BILLION BOOKS PER DAY

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33 Alexandrov LB et al., Science Nov 4;354(6312):

34 Recent Evidence Shows that Epigenetic Changes are Also Important in Causing Cancer GENETIC EPIGENETIC Example: Replication errors Example: Chromatin modification errors X X Altered DNA sequence Altered DNA/mRNA/proteins Oncogenesis Altered chromatin structure Altered levels of mrna/proteins Tumor

35 Epigenetics Can Cooperate With Genetic Mutations to Promote Oncogenesis GENETIC EPIGENETIC Oncogene function Tumor suppressor function Oncogene levels Tumor suppressor levels Oncogenesis Tumor

36 Epigenetics Play Important Roles in Normal Cellular Development and in Cancer EPIGENETICS Normal epigenetic mechanisms Deregulated epigenetic mechanisms Normal differentiated cells, e.g. embryonic cells, hematopoetic cells Malignant progenitor cell Tumor Epigenetic mechanisms can regulate genes involved in differentiation, cell cycle, and cell survival Deregulation of epigenetic mechanisms results in aberrant gene expression, which can lead to cancer

37 Balance of Histone etylation is a Key Factor in Transcriptional Regulation in Normal Cells HAT TF HISTONE ACETYLATION TF Deacetylated Histones Closed chromatin Transcription factors cannot access DNA Gene expression etylated Histones Open chromatin Transcription factors can access DNA Gene expression

38 Balance of Histone etylation is a Key Factor in Transcriptional Regulation in Normal Cells HAT TF HISTONE ACETYLATION TF Deacetylated Histones Closed chromatin Transcription factors cannot access DNA HISTONE DEACETYLATION HDAC HDAC etylated Histones Open chromatin Transcription factors can access DNA Gene expression Gene expression

39 Imbalanced Levels of Histone etylation in Cancer Deregulate Gene Expression HAT HISTONE ACETYLATION TF TF HISTONE DEACETYLATION Deacetylated Histones Closed chromatin Transcription factors cannot access DNA HDAC etylated Histones Open chromatin Transcription factors can access DNA Increased HDAC activity or decreased HAT activity may result in aberrant gene expression, contributing to cancer

40 Increased HDAC tivity Can Alter Gene Expression and Result in Cancer TF Gene expression HDAC Increased HDAC activity, which has been associated with certain tumors, can alter expression of genes involved in normal cell development, resulting in: Loss of cell-cycle arrest Inhibition of differentiation Cell growth and proliferation Evasion of apoptosis Migration and metastasis Cell nucleus

41 Evidence of Aberrant Epigenetic States in Cancer Genes involved in DNA methylation and histone modifications are deregulated in multiple tumor types Over 40 genes have been shown to be silenced by DNA hypermethylation in a variety of tumor types p15 (CDKN2B), MLH1, BRCA1, BCL2L11, RB, AR, APC

42 Therapeutic Targeting of Both Histone and DNA Modifications Can Synergize DNMT TF Gene expression DNMT Inhibitor DAC Inhibitor Cell-cycle arrest Differentiation Growth control Apoptosis HDAC Since DNA methylation and histone deacetylation can co-operate to silence tumor suppressors, inhibition of both DNMT and HDAC activities can synergize to restore expression of silenced genes Adhesion Cell nucleus

43 CLASSIFICATION OF EPIGENETIC DRUGS

44 Summary Epigenetic processes regulate gene expression and cell behavior Epigenetic changes leading to gene silencing are central to the pathogenesis of hematologic and solid malignancies Histone deacetylation and DNA methylation represent two epigenetic modifications with clinical relevance to oncogenesis Therapeutic targeting of epigenetic processes can restore expression of genes that are critical for the control of normal cell growth HDAC inhibitors display synergy with DNA demethylating agents to inhibit tumor growth

45 Protocols

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