1. Assessing functional consequences of epigenetic modifications An Data Analysis Activity for Students This teacher slide set was created by Dana Haine, MS, of the UNC Superfund Research Program, which is funded by the National Institute of Environmental Health Sciences (P42ES005948).

2. Is there is a relationship between prenatal exposure to arsenic and functional epigenetic changes to newborn DNA ? Does exposure + epigenetic changes = changes to gene expression? Assessing functional consequences of epigenetic modifications

3. Dr. Rebecca Fry Studies the link between prenatal exposure to arsenic and cadmium and newborn health. LinkLink to Video describing her research.

4. Research Questions in the Fry Lab What is the relationship between prenatal arsenic exposure and changes to gene expression? Are any of the genes that are altered in association with arsenic controlled by the epigenetic mechanism DNA methylation?

5. How were these questions investigated? 38 newborns – Prenatal arsenic exposure – DNA Methylation Profiles – RNA (Gene expression) Profiles

6. Microarray analysis was used to determine which genes exhibited differential expression The microarray works by first extracting DNA or RNA and hybridizing different pieces representing different genes to a transcript It allows us to measure how many copies of each gene is expressed This can tell us whether there is no, some or a lot of RNA transcript present

7. 334 U-tAs-associated transcripts N=38 subjects with range of arsenic exposure, cord blood (vein) isolated N=224 N=110 A heatmap is a way to visualize data trends from microarray U-tAs (µg/L)

8. Conclusions from Heat Map There are 334 genes that show a change in transcript profiles in association with arsenic exposure in the 38 babies. Some gene expression profiles increase with increasing arsenic exposure and some gene expression profiles decrease with decreased arsenic exposure These changes suggest that prenatal arsenic exposure does affect a baby’s gene expression profile

9. Are any of the genes whose expression is altered in association with arsenic controlled by DNA methylation?

10. The traditional view of DNA methylation

11. What would you expect to find? ? Methylation Level Level of gene expression

12. What would you expect to find?

13. What did we see when looking at individual genes? DNA Methylation Gene Methylation RNA Transcript Gene Expression

14. Conclusions 2,919 genes exhibited differential methylation in response to arsenic exposure 334 gene exhibited corresponding changes in gene expression (mRNA transcripts) Only 16 genes exhibited a significant linear relationship between methylation and gene expression Seven of these genes were related to important birth outcomes including gestational age and head circumference

15. For most genes, researchers found no correlation between gene expression and methylation

16. This pattern is present across the entire genome!