The Emerging Role of Epigenetics in Human

Diseases

David P. Gardner, Ph.D.Professor of Biochemistry

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.

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

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)

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.

Another Familiar Epigenetic Case

Prader-Willi Syndrome Angelman Syndrome

Chromosome 15 imprinting

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.

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.

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Germ Cells

Somatic Cells

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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).

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.

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. Kaati G, Bygren LO, Edvinsson S. Eur J Hum Genet. 2002, 10:682-8.

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).

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.

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.

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).

Cytosine Methylation

Methylation of cytosine occurs at CpG dinucleotides.

Often located just upstream of genes (promoter regions).

Associated with attenuation of expression of nearby genes.

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.

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.

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.⬆

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.⬆

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 CpG dinucleotides and silencing of the FMR1 gene.

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).

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.⬆

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Combination Therapy

Phase III Clinical Trial

Vorinostat plus cytarabine and idarubicin.

85% remission rate after initial treatment.

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.

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.”

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.

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.

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.

Epigenetics and Osteopathic Manipulation

Is it plausible that osteopathic manipulation could influence gene expression through modulation of epigenetic tags on treated tissue?

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.

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 Clin Haematol. 25, 427-435.

Fraga, M.F. et al. 2005. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci 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.