Epigenetics and Functional Genomics: Mechanisms, Environment, and Health Impacts

Conceptual Definition: Epigenetics is the mechanism that allows for cell identity. While every cell in the human body contains the entire genome, cells distinguish themselves by which genes are accessible and which are locked away.
Cellular Comparison: Keratinocytes found in skin and osteoclasts found in bone contain identical genetic sequences. The body prevents skin cells from transforming into bone cells by utilizing a "massive genetic padlock" to lock away the instructions (genes) needed to create bone cells. Furthermore, the "keys" for these padlocks, known as transcription factors, are themselves locked away.
Gene Silencing Statistics: Cells carry out specific functions by expressing only a subset of their genes. In any given specific cell, approximately $80-90\%$ of the genome is silenced.
Differentiation Example (Fibroblasts to Adipocytes):
- Fibroblast-like cells exhibit active GATA 1 and GATA 2 genes but inactive PPAR $\gamma$ and CD36 genes.
- Fat cells (adipocytes) exhibit inactive GATA 1 and GATA 2 genes but active PPAR $\gamma$ and CD36 genes.
- During differentiation, GATA 1 and 2 are not removed from the genome; rather, the stretch of DNA where they reside becomes tightly locked.

Mechanism: Methylation occurs at the lysine residues of the histone tail. This process is coordinated by Histone Methyl-transferases (HMTs).
Function: It works by recruiting proteins to the chromatin that either zip or unzip the structure. It can be associated with either gene activation or repression.
Specific Markers:
- $H3K4me3$ : Associated with open chromatin located at the transcription start site (the "starting line") of a gene.
- $H3K27me3$ : Associated with closed, repressed chromatin.
Clinical Significance (Alzheimer's Disease): Epigenetic drift in Alzheimer's patients causes methylation at $H3K4$ to drop. This effectively "locks" the genes required for memory formation (Wang, Oelze et al. 2008).

Mechanism: This process generally associates with gene activation. It requires Histone Acetyl-transferases (HATs).
Biochemical Action: Acetylation works by neutralizing the positive charge of the lysine residue, which allows the chromatin structure to loosen.
Clinical Significance (Rubinstein-Taybi Syndrome): This syndrome is associated with mutations in HAT proteins. Because the HATs are non-functional, the chromatin remains locked, and the genes required for normal neural development cannot be switched on (Petrif, Giles et al. 1995).

Mechanism: DNA methylation occurs on the chromosomal DNA itself and is generally associated with gene repression.
Coordination: It is managed by DNA Methyl-transferases.
Action: It physically blocks gene expression or recruits proteins that zip the chromatin up more tightly.
The "Epigenetic Scar": DNA methylation can act as a permanent mark on the genome. For example, methylation at specific sites can indicate whether an individual is a current smoker, a former smoker, or has never smoked.

DNase-seq:
- Uses the DNase enzyme to cut DNA at random locations.
- The enzyme can only cut DNA that is open and accessible.
- This technique reveals DNase I Hypersensitivity Sites (DHS), which identifies regions considered to be transcriptionally active.
Chromatin Immuno-precipitation seq (ChIP-seq):
- The process begins by "freezing" the chromatin and any attached proteins using formaldehyde.
- Specific antibodies are employed to capture DNA-bound proteins of interest.
- After the proteins are removed, the attached DNA is sequenced.
- This can be used to isolate genes located near methylated histones.

Study Design: Researchers examined offspring from mother rats exhibiting two distinct behaviors:
1. High licking/grooming (LG) behavior.
2. Low LG behavior.
Observations:
- Rats raised by high LG mothers were more relaxed and eventually became high LG mothers themselves.
- Rats raised by low LG mothers exhibited higher anxiety.
Biological Explanation: The behavioral differences were attributed to high methylation and low histone acetylation surrounding the glucocorticoid receptor (GR) gene. In the anxious animals, the GR gene was effectively switched off (Weaver, Cervoni et al. 2004).

Exercise: Triggers global hypomethylation at genes specialized for fat oxidation and muscle repair. This change typically persists for several days, but in athletes, these genes may stay switched on permanently.
Smoking: Alters the methylome (the global pattern of methylation). It leaves epigenetic scars associated with malignancies. These changes are dose-dependent; many recover after years of cessation, depending on the individual's smoking history.
Obesity: Persistent obesity is associated with epigenetic changes in genes linked to fat cell function. A controversial theory known as "obesogenic memory" suggests these changes might be irreversible.

Definition: Obesogens are exogenous chemicals that cause fat tissue to accumulate either directly or indirectly.
Context: Obesity prevalence is rising at a rate that lifestyle changes alone cannot explain. Mozaffarian (2022) noted that there is no clear explanation for the current obesity epidemic.
Tributyltin (TBT): A potent obesogen (now banned) formerly used as an antifouling agent.
- TBT reduces global methylation in fat cells.
- Cells exposed to TBT differentiated at higher rates than control groups.
Quantitative Data (Bastos Sales, Kamstra et al. 2013):
- Regression Equation: $y = -0.0555x + 4.0185$
- Correlation: $R^2 = 0.821$
- Example: Differentiation ranges from $0$ to $50\%$ while methylation ranges from approximately $3.7\%$ to $4.05\%$
- Chemicals involved in studies include TBT, TRO, BDE-47, BPA, TCDD, and HCB.

Hormonal Interference: Chemicals leaching from plastics interfere with hormonal signaling in fat cells. They are associated with increased waist circumference and insulin resistance in humans.
Molecular Mechanisms: Exposure to PACs is linked to relaxed chromatin near the promoter for $PPAR\gamma$ (the master regulator of fat cell development) and differential Histone 3 methylation across genes related to fat cell development (Hurst and Waxman 2003).
The Obesogenic Floodgate Problem:
- Phthalates "prime" Adipose-derived Stem Cells (ASCs) for obesity.
- Prenatal exposure is linked to obesity later in life.
- Phthalates may physically switch on fat cell development by interacting with proteins involved in gene expression.
- The effect is amplified because it exerts pressure on a population that is already significantly more susceptible to obesity.
- Research is difficult because PAC exposure is ubiquitous, potentially even reaching uncontacted tribes.

Task 1: Comparison of DNase Seq and RNA Seq reads across the $CYP1B1$ gene in lung cells taken from two different patients.
Task 2 (Chromatin Accessibility Scores): This score indicates relaxed chromatin (heterochromatin):
- <1: Gene suppression.
- $\approx 1$ : Neutral state.
- >1: Gene expression.