UNC-Chapel Hill Superfund Research Program Assessing biological responses to contaminants An introduction to epigenetics & the exposome

Same genes, different phenotypes How? NOVA’s A Tale of Two Mice: Chapter 1

Lesson: DNA Wrap-Packaging Matters Learning Objectives Define the make-up of chromatin Define epigenetics Describe DNA methylation as a means of inhibiting transcription Evaluate impact of environmental agent on gene expression (in mice)

(between adjacent nucleotides) (consists of a base, a 5-carbon sugar, and a phosphate group) Hydrogen Bonds (between bases) Phosphodiester Bond (between adjacent nucleotides) Decondensed Chromatin Condensed http://commons.wikimedia.org/wiki/File:Chromosome.gif

Genome vs epigenome Genome Epigenome Genes Accessibility to genes Hardware Static Epigenome Accessibility to genes Software Plastic

What is Epigenetics? Refers to changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence. “The study of stable alterations in gene expression potential that arise during development and cell proliferation” (Nature). Enables a cell/organism to respond to its dynamic external environment during development and throughout life! Carey page 90 quote

The Epigenome at a Glance Genetic Science Learning Center, University of Utah

NOVA’s Ghost in Your Genes (2006) Now it appears that our diets and lifestyles can change the expression of our genes. How? By influencing a network of chemical switches within our cells collectively known as the epigenome. NOVA’s Ghost in Your Genes (2006)

Epigenetic Mechanisms DNA Methylation Histone Modification Small, non-coding RNAs (e.g., micro RNA)

2. DNA methylation The addition of methyl groups to DNA, mostly at CpG sites, to convert cytosine to 5-methylcytosine. 5’ 3’ SAM C G T A C A C G A C A C G A T G C A T G T G C T G T G C T A 3’ 5’ represent potentially heritable changes to the genome

DNA methylation is preserved during DNA replication Stable epigenetic modification; enables differentiated cells to stay differentiated

DNA methylation leads to gene silencing Target gene expressed in normal cell Normal State RNA pol TF Target gene expressed Promoter Target gene inactivated by DNA methylation Epigenetic Modification TF X Target gene not expressed Promoter DNA methylation at promoter regions can impede target gene expression CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. *"CpG" stands for cytosine and guanine separated by a phosphate (—C—phosphate—G—), which links the two nucleosides together in DNA.

1) Changes to DNA Methylation leads to gene silencing Target gene expressed in normal cell e.g., tumor suppresor gene Normal State RNA pol TF Target gene expressed Promoter Target gene inactivated by hypermethylation Disease State arising from Epigenetic Modification TF X Target gene not expressed Promoter DNA methylation at promoter regions can impede target gene expression CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. *"CpG" stands for cytosine and guanine separated by a phosphate (—C—phosphate—G—), which links the two nucleosides together in DNA.

2) Changes to DNA Methylation leads to gene activation Target gene silenced by methylation e.g., oncogene Normal State TF X Target gene not expressed Promoter Target gene activated by hypomethylation or demethylation Disease State arising from Epigenetic Modification RNA pol TF Target gene expressed Promoter DNA methylation at promoter regions can impede target gene expression CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. *"CpG" stands for cytosine and guanine separated by a phosphate (—C—phosphate—G—), which links the two nucleosides together in DNA.

HOW did these mice become different? NOVA’s A Tale of Two Mice Chapters 2 &3

In 2003, a research team at Duke discovered HOW changing the diet of Agouti yellow female mice resulted in genetically identical offspring with altered coat color distribution and disease susceptibility. http://randyjirtle.com/index.php?page=2

? Agouti mouse model Avy = Agouti Viable Yellow Dominant allele; paternally inherited Allele is always expressed a = mutant version of agouti gene Genetically identical except for agouti gene ?

Genetically Identical Siblings Epigenetic Variation From the following article: Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) http://www.nature.com/nrg/journal/v8/n4/fig_tab/nrg2045_F2.html

Michael Skinner, A New Kind of Inheritance (2014) The most important effect of epigenetic marks – maybe their reason for existing – might be to wildly expand the number of variant individuals in a population. Michael Skinner, A New Kind of Inheritance (2014)

In this mouse model, a shift in coal color distribution indicates changes in epigenetic modifications. From the following article: Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) http://www.nature.com/nrg/journal/v8/n4/fig_tab/nrg2045_F2.html

TIME MAGAZINE, PERSON OF THE YEAR 2007 Dr. Jirtle's pioneering work in epigenetics and genomic imprinting has uncovered a vast territory in which a gene represents less of an inexorable sentence and more of an access point for the environment to modify the genome. TIME MAGAZINE, PERSON OF THE YEAR 2007

Experiment: Does a maternal diet with soy influence expression of the agouti gene? Female mice were fed genistein, the major phytoestrogen in soy, before and during pregnancy.

Shift in coat color distribution… Genetically identical offspring exhibited varying coat colors.

23% 50% From the following article: Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) Environmental epigenomics and disease susceptibility Randy L. Jirtle and Michael K. Skinner Nature Reviews Genetics 8, 253-262 (April 2007) http://www.nature.com/nrg/journal/v8/n4/fig_tab/nrg2045_F2.html

Experimental results

Genetic Science Learning Center, University of Utah Conclusions Read the authors’ conclusions and with a partner discuss how these conclusions could be relevant for humans.

Genetic Science Learning Center, University of Utah Conclusions Our diet, especially during pregnancy, can influence our epigenome and ultimately determine our susceptibility to cancer and disease.

Michael Skinner, A New Kind of Inheritance (2014) But nurture matters too. Many of the contingencies of life- what we eat, what pollutants are in our environment, how often we are stressed – affect how the genes operate. Michael Skinner, A New Kind of Inheritance (2014)

Epigenetics and fetal development “Epigenetics is being considered an important mechanism through which various stressors can operate. Many lines of evidence point to their being a special concern during rapid fetal development.” Frederica Perera, Columbia Center for Children’s Environmental Health

Inheritance of Epigenetic modifications Multigenerational Inheritance from parent to progeny Meiotic or transgenerational Inheritance by F3 offspring (rare; not documented in human studies to date) Vinclozolin exposure and rats

Rule out the possibility of genetic changes; and, To demonstrate transgenerational epigenetic inheritance researchers must: Rule out the possibility of genetic changes; and, Show that the epigenetic effect can pass through enough generations (F3 or great grandchild) to rule out the possibility of direct exposure. Genetic Science Learning Center, University of Utah

Contact Information Superfund Research Program University of North Carolina at Chapel Hill http://www.sph.unc.edu/srp/ Resources > For educators Research Translation Core Dana Haine dhaine@unc.edu (919) 843-5735

If time permits…

Imprinting Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner It is an inheritance process independent of the classical Mendelian inheritance. The first naturally occurring example of an imprinted gene was the discovery of imprinting in the IGF-2 gene in mice in 1991, and currently about ~150 imprinted genes have been identified in mice and humans. http://ls-space.blogspot.com/2008/07/047-evolutionary-origin-of-mammalian.html

DNA methylation & imprinting Imprinted genes are inactive due to DNA methylation. For imprinted genes, only one gene copy is active http://learn.genetics.utah.edu

DNA methylation & imprinting Imprinted genes are protected from reprogramming that occurs in zygote http://learn.genetics.utah.edu

The Environment & Imprinted Genes Environmental signals can affect the imprinting process during gamete development Imprinted genes in the adult are sensitive to environmental signals (since only one gene copy is active)

Imprinting: an inheritance process independent of Mendelian inheritance

Why Imprint? The Genetic Conflict Hypothesis Many imprinted genes are involved in growth and metabolism. Often maternally and paternally imprinted genes work in the very same growth pathways. This conflict of interest sets up an epigenetic battle between the parents -- a sort of parental tug-of-war. Randy L. Jirtle and Jennifer R. Weidman, 2007 Pg 131 Carey

Acetylation is the most highly studied Histone modifications are more plastic than DNA methylation,

3. Non-protein coding RNAs short, non-coding RNAs e.g., microRNAs partially complementary to one or more mRNA molecules post-transcriptional gene regulation, typically down regulation Figure: http://www.journalofanimalscience.org/content/87/14_suppl/E21.full http://www.nature.com/nrg/multimedia/rnai/index.html 43

1. Histone modification If the way that DNA is wrapped around the histones changes, gene expression can change. Histone Modification Plastic; enables cell to respond to stimuli, allows for fine tuning of gene expression. http://ehp.niehs.nih.gov/wp-content/uploads/2012/09/WP_Focus_2_Title_web.png

Post-translational histone modifications Although histone modifications occur throughout the entire sequence, the unstructured N-termini of histones (called histone tails) are particularly highly modified. Carey: more than 50 different epigenetic modifications to histones have been identified (2012) – pattern of modifications is called a histone code.