The Emerging Role of Epigenetics in Human Diseases

1. The Emerging Role of Epigenetics in Human Diseases • David P. Gardner, Ph.D. • Professor of Biochemistry

2. Objectives • Provide a working definition of epigenetics and contrast an epigenetic change with a mutation. • Contrast the normal process of genomic imprinting with abnormal changes in epigenetic tags seen in several diseases described. • Describe evidence that nutritional status can influence the epigenetic profile of later generations. • Illustrate and describe epigenetic tags involving cytosine methylation and histone acetylation. • Describe the mechanism of action of Vorinostat. • Interpret the divergence of epigenomes of identical twins with respect to potential difference in disease presentation between twins.

3. Epigenetics • Epigenetics literally means ‘above’ the genetics. • Has had multiple definitions over time. • 2008 Cold Spring Harbor Epigenetics meeting: • “An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.” • Alterations in the DNA sequence = mutations

4. Epigenetic Research • The number of publications in the field has increased dramatically in the last 10 years. • Genetic Engineering and Biotechnology News Feb 1, 2013 (Vol. 33, No. 3)

5. Epigenetic Effects • The effects of epigenetics have been known for many years. • Lyon Hypothesis from 1961. • Renamed the Lyon Law in 2011. • Inactive X chromosome is heavily epigenetically modified.

6. Another Familiar Epigenetic Case • Chromosome 15 imprinting • Prader-Willi Syndrome • Angelman Syndrome

7. Angelman/Prader-Willi • Commonly referred to as genomic imprinting. • Imprinting is not the cause of these syndromes but is responsible for the unique presentation of these diseases.

8. Germ Cells * Genomic Imprinting • With genomic imprinting, it is thought that the maternal or paternal imprint is erased with each succeeding generation (meiotic division). • A male receives a maternally imprinted and paternally imprinted chromosome 15 but will always transmit a paternally imprinted chromosome 15. • Note that the maternal/paternal imprinting is heritable through mitosis. Somatic Cells * * * * * *

9. Genomic Imprinting • Importantly, X-inactivation and genomic imprinting are normal processes. • Much of the recent research has analyzed when the process of epigenetics is altered from normal. • This has involved the study of changes within somatic cells in disease. • It has also involved the study of changes within the germ cells (heritable epigenetics).

10. Heritable Epigenetics • Evidence suggests that environmental information could be propagated through meiosis. • Studies of Dutch famine of 1944. • Famine during last two trimesters of pregnancy: • 8-9% decrease in child’s birth weight (SGA). • Offspring of these SGA children tended to be normal size. • Famine early in pregnancy but not late: • Normal size infants were born. • Offspring of these non-SGA children exhibited high rate of SGA.

11. Överkalix Study • Retrospective study conducted in Överkalix, Sweden. • Divided population into three cohorts: Born 1890 Born 1905 Born 1920 • Assessed each cohort for access to food during slow growth period (SGP) of adolescence (8-10 girls, 9-12 boys). • Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. KaatiG, Bygren LO, EdvinssonS. EurJ Hum Genet. 2002, 10:682-8.

12. Överkalix Study Results • When the father (P=0.05) was exposed to a famine during his SGP, his offspring exhibited protection against cardiovascular causes of death. • Paternal grandmother exposure to famine also showed a trend (P=0.11) towards similar protection in grandchildren. • If the paternal grandfather lived through a famine during his SGP it tended to protect grandchildren from diabetes (P=0.09).

13. Överkalix Study Results • If the paternal grandfather had an abundance of food during their SGP, their grandchildren had a four-fold increased risk for death of diabetes mellitus. • One mechanism to explain these results is transmission of epigenetic markers that were influenced by the environment of the parent. • Effect on grandchildren suggests the markers are maintained through multiple generations.

14. Lamarkism? • Jean Batiste Lamark (1744-1829) • Inheritance of acquired characteristics. • Largely discounted with Darwin’s publication of Origin of Species and the rediscovery of work of Mendel. • Recent work in epigenetics suggest Larmark may have been correct to some degree.

15. Molecular Basis of Epigenetics • Two primary mechanisms identified. • Methylation of cytosine nucleotides in DNA • Posttranslational modification to histone proteins. • Includes acetylation, methylation and phosphorylation • A third proposed mechanism involves expression of small interfering RNAs (siRNA).

16. Cytosine Methylation • Methylation of cytosine occurs at CpGdinucleotides. • Often located just upstream of genes (promoter regions). • Associated with attenuation of expression of nearby genes.

17. Histone Modification • Histones are the proteins that organize the genetic material. • Have a high percentage of basic amino acids, which gives histones an overall positive charge. • Positively charged amino acids associate with the overall negative charge of the DNA.

18. Histone Modification • Most histone modification occurs on the extended tails of histone proteins. • Modifications influence the association of histones with the DNA and patterns of gene expression. • Best studied modification is histone acetylation.

19. Histone Acetylation • Two enzyme types involved in histone acetylation • HAT: histone acetyltransferase • HDAC: histone deacetylase • Note that acetylation eliminates the positive charge from the amino acid. • It is thought that this changes the chromatin conformation to a form more open to transcription. • ⬆ acetylation = ⬆gene expression.

20. HAT/HDAC and Hydrophobic Hormones • It is thought that hydrophobic hormones like thyroid hormone and glucocorticoid influence gene expression by binding to either HDAC or HAT proteins. ⬆ acetylation = ⬆gene expression.

21. Epigenetic Errors • Fragile X syndrome is most commonly caused by a CGG trinucleotide repeat expansion in the 5’ region of the FMR1 gene. • Unaffected individuals have 6-50 CGG repeats. • >200 CGG repeats is seen in individuals with fragile X. • >200 CGG repeats is correlated with hypermethylation at CpGdinucleotides and silencing of the FMR1 gene.

22. Epigenetics and Cancer • DNA repair is a critical process to maintain genomic fidelity. • Loss of DNA repair is thought to be a major contributor to the development of cancer. • Epigenetic changes involving DNA repair genes are thought to be a major early step in cancer progression. • ~13% of sporadic breast cancers and 5-30% of ovarian cancers present with hypermethylation of the BRCA1 gene. • 40-90% of sporadic colorectal cancer has hypermethylation of the MGMT gene (O6-methylguanine methyltransferase).

23. Therapies Targeting Epigenetic Errors • In contrast to mutations, epigenetic changes can be reversed. • Are there therapies that influence epigenetic patterns? • Yes • Vorinostat (trade name Zolinza) approved by FDA for cutaneous T cell lymphoma in 2006. • Vorinostat is a histone deacetylase inhibitor. • ⬆ acetylation = ⬆gene expression. X

24. Combination Therapy • Phase III Clinical Trial • Vorinostat plus cytarabine and idarubicin. • 85% remission rate after initial treatment.

25. Our Epigenome • If epigenetic markers are dynamic and respond to environmental influences, do they change over time? • Evidence suggests the answer is yes. • Twin studies have been highly informative for this question.

26. Author Statement • “We found that, although twins are epigenetically indistinguishable during the early years of life, older monozygous twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene-expression portrait.”

27. Chromosomal Level • Comparative genomic hybridization for methylated DNA • Yellow = similar chromosome methylation pattern between twins. • Red = regions of hypomethylation in one twin compared to the other. • Green = regions of hypermethylation in one twin compared to the other.

28. Epigenomic Alterations • If the epigenome changes as we age, what kinds of things can induce these changes? • Very active area of current research. • Some interesting findings: • Fear conditioning induces changes in DNA methylation in the brain derived neurotrophic factor (BDNF) gene promoter region in hippocampal neurons of rat brains.

29. Epigenomic Alterations • In rats, social deprivation during the 1st postnatal week triggers changes in DNA methylation across the BDNF gene. • This was later associated with decreased BDNF gene expression in the prefrontal cortex of adult experimental animals. • A schizophrenic-type state can be induced in mice when they are chronically given l-methionine (substrate for methyltransferase enzymes). • Studies with cocaine exposure suggest that the drug induces acetylation of the BDNF gene histones that is transmittable to future male offspring.

30. Epigenetics and Osteopathic Manipulation • Is it plausible that osteopathic manipulation could influence gene expression through modulation of epigenetic tags on treated tissue?

31. Summary • Epigenetic traits are heritable phenotypes resulting from changes in chromosomes that do not involve changes in DNA sequence. • Scientific and medical interest in epigenetics has increased dramatically in recent years. • Two prominent epigentic mechanisms involve DNA methylation (gene silencing) and histone acetylation (gene activation). • Errors in epigenetic patterns can influence the presentation of human diseases including cancer. • The epigenome changes as we age and can be influenced by the environment. • Drugs that influence the epigenome represent a major area of current research.

32. References • Berger, S.L. et. al. 2009. An operational definition of epigenetics. Genes Dev.23, 781-783. • Kaati, G. et. al. 2002. Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Eur. J. Hum. Genet. 10 682-688. • Esteller, M. et. al. 2000. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst. 92, 564-569. • Shen, L. et. al. 2005.MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst. 97, 1330-1338. • Garcia-Manero, G. 2012. Can we improve outcomes in patients with acute myelogenous leukemia? Incorporating HDAC inhibitors into front-line therapy. Best Pract Res ClinHaematol.25, 427-435. • Fraga, M.F. et al. 2005. Epigenetic differences arise during the lifetime of monozygotic twins. ProcNatlAcadSci U S A. 102,10604-10609. • Roth, T.L. et. al. 2009. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biol Psychiatry. 65 760-769. • Vassoler, F.M. 2013. Epigenetic inheritance of a cocaine-resistance phenotype. Nat. Neurosci. 16, 42-47.