Lecture 6

Epigenetics Research Methods and Analysis

Introduction to Epigenetics

  • Key Topics Covered:

    • Epigenetic mechanisms

    • Aging

    • Disease mechanisms

    • Environmental health

    • Cell biology research areas

  • Understanding Epigenetics:

    • Deepens comprehension of cellular function

    • Evaluates environmental effect on human biology

    • Explores disease mechanisms

    • Investigates aging processes

  • Importance of Epigenetic Research:

    • Critical for diagnosis, prevention, and treatment of diseases.

Crosstalk Between Epigenetics and Genetics

  • Overview of Crosstalk:

    • Epigenetic processes influence gene expression patterns.

    • Alterations in epigenetic modifications can stem from:

    • Genetic mutations in epigenetic modifiers

    • Availability of chemical groups.

  • Implications:

    • Abnormal crosstalk can lead to diseases such as cancer.

Techniques in Epigenetic Studies

  1. DNA Methylation Analysis:

    • Techniques include bisulfite conversion and DNA analysis, and epigenetic-wide association studies (EWAS).

  2. Chromatin Accessibility:

    • Assay of transposase accessible chromatin sequencing (ATAC-seq).

  3. Histone Modifications Analysis:

    • Chromatin immunoprecipitation (ChIP) techniques.

  4. Microscopy and Chromatin Imaging:

    • Utilized for examining chromatin structure.

  • Goal of Techniques:

    • Investigate epigenetic modifications (DNA methylation or histone modifications).

    • Detect abnormalities caused by genetics or environmental factors.

DNA Methylation Analysis

Multistep Analysis

  • Levels of Analysis:

    • DNA analysis can occur at three genomic resolutions:

    • Global

    • Regional

    • Base-pair

    • Epigenetic-wide association study (EWAS) enables comprehensive analysis of methylation alterations.

Bisulfite Conversion Process

  • Importance:

    • Cornerstone of DNA methylation analysis since early 1990s.

  • Key Principles:

    • Sodium bisulfite converts unmethylated cytosines to uracil, leaving 5-methylcytosines unchanged.

    • Enables single-nucleotide-resolution mapping of DNA methylation.

    • Non-methylated cytosines deaminate to uracil while methylated ones remain intact in CpG dinucleotides.

Requirements for Accurate Methylation Analysis

  • Complete Conversion of DNA:

    • Incomplete conversion leads to misinterpretation of unmethylated cytosines as being methylated.

  • DNA Denaturation:

    • DNA must be fully denatured to expose cytosine residues to sodium bisulfite.

  • Control Variables:

    • Proper management of salt concentration, temperature, and incubation time to achieve complete denaturation.

Gene and Genome Level Analysis

  • At the Gene Level:

    • Analyzing methylation of specific genes.

  • At the Genome Level:

    • Utilizing PCR after bisulfite treatment for genomic sequencing of converted DNA.

  • Polymerase Chain Reaction (PCR):

    • A DNA amplification technique using sequence-specific primers.

Methylation-Specific PCR (MSP)

  • Classical Method:

    • Uses two sets of primers:

    • One for amplifying unmethylated DNA.

    • Another specific for methylated DNA segments.

    • Analysis conducted through gel electrophoresis after two PCR reactions per sample.

Practical Example: Prader-Willi Syndrome (PWS)

  • Characteristics of PWS:

    • Complex disease linking genetic and epigenetic factors.

    • Involves chromosomal region 15q11-q13 with multiple imprinted genes, alternative splicing, gene duplications, and copies regulating imprinting.

  • Diagnostic Testing:

    • Karyotyping followed by further molecular analysis is needed to confirm diagnosis through epigenetic methylation methods.

Case Study Discussion: Identical Twins and Epigenetics

Case Summary

  • Subjects: Elise and Shannon, identical twins with similar childhood experiences.

    • Shannon diagnosed with schizophrenia at age 19.

    • Elise investigates her own disease risk after Shannon’s diagnosis, as schizophrenia has familial tendencies.

Key Points About Schizophrenia

  • Genetic Contribution:

    • 50% of the disorder’s causes attributed to genetic factors.

  • Environmental Contribution:

    • Other 50% is influenced by environmental factors including stress and relationships.

    • Environmental changes may affect DNA structure and packaging, impacting trait development.

DNA Methylation in Twins

  • Research Overview:

    • Differential DNA methylation observed between monozygotic (identical) twins, highlighting the impact of age and environment.

    • Use of amplified inter-methylated sites (AIMS) to detect this methylation.

REELIN Signaling and Schizophrenia

  • REELIN:

    • A secreted glycoprotein linking to several receptors involved in neuron migration and brain development.

    • Variations in REELIN expression may correlate with schizophrenia vulnerability.

Class Discussion and Techniques for Confirmation

  • Discussion Topics:

    • Techniques to confirm low risk of schizophrenia in Elise.

    • Analyzing methylation of the RELN promoter/enhancer and appropriate methodologies.

Summary of DNA Methylation Analysis

  • Methods of Analysis:

    • At gene level: specific gene methylation assessment.

    • At genome level: employing PCR for bisulfite-converted genomic sequencing.