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. Assessing functional consequences of epigenetic modifications
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?
We are aiming to answer this question, but what does this question mean? And how do we get here?

3. Dr. Rebecca Fry
Studies the link between prenatal exposure to arsenic and cadmium and newborn health.
Link to Video describing her research.
Uses systems toxicology to understand the molecular mechanisms by which early-life exposures to metals such as cadmium and arsenic are associated with long-term health effects in humans.

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?
So In our work we had two major research questions we wanted to answer
X and X
We’re going to discuss the experimental design we used to determine the answers to these questions

5. How were these questions investigated?
38 newborns
Prenatal arsenic exposure
DNA Methylation Profiles
RNA (Gene expression) Profiles
We analyzed DNA and RNA from samples from the BEAR cohort using umbilical cord blood collected at birth
from 38 newborns.
The newborns had mothers that had a range of arsenic exposure as identified by levels of iAs in maternal urine.
RNA transcript levels were assessed using a microarray analysis that gave us gene expression for around 20,000 genes.
Then DNA Methylation was analyzed using a separate microarray analysis that assessed 450,000 CpG sites.

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
So once we have the microarray data we can ask the question “which gene expression levels show a relationship to changes in arsenic exposure”
Once we have determined what genes change in relationship to arsenic exposure we can display these in a heatmap

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

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
So given the traditional view of DNA methylation we would say that any of the genes that increase in expression in association with arsenic exposure should have fewer methyl groups and genes that show increased expression in response to arsenic exposure should have fewer methyl marks

11. What would you expect to find?
Level of gene expression
What we would expect to see when we graph gene expression vs. gene methylation would be that there was a linear relationship showing that as methylation increased, gene expression decreased
Methylation Level

12. What would you expect to find?
What we would expect to see when we graph gene expression vs. gene methylation would be that there was a linear relationship showing that as methylation increased, gene expression decreased

13. What did we see when looking at individual genes?
RNA Transcript
Gene Expression
So even when we look at the 239 differentially expressed and differentially methylated genes, only 16 of them actually displayed a relationship.
The rest all had null associations.
DNA Methylation
Gene Methylation

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
Gestational age has been associated with slower growth, increased incidence of illnesses (cold, flu, etc. ), and an increased likelihood of developing asthma
Head circumference have been associated with autism spectrum disorders and other neurologic outcomes

15. For most genes, researchers found no correlation between gene expression and methylation
But as you saw earlier today this was not the case. When we looked at the 239 genes that were on both platforms we found that there was no relationship between differential methylation of a gene and differential expression of a gene

16. This pattern is present across the entire genome!
We also saw this trend hold for the whole genome, not just genes associated with arsenic.
For genes that are highly expressed we would expect them to have low methylation values and for genes that are lowly expressed we would expect high methylation values.
What we found was that this general trend is somewhat apparent as evidenced by the peask on the two sides, BUT there are lots of genes with middle levels of expression
And there are genes that have high expression and high methylation and low expression and low methylation.