Epigenetics Study Guide

Flinders University Epigenetics Study Notes

Introduction to Epigenetics

  • Overview: The video aims to cover the role of epigenetics in our cells, defining epigenetic modifications and introducing the two main types that will be studied.
  • Communication Note: Material reproduced under section 113P of the Copyright Act 1968, indicating copyright protections.

Learning Objectives

  • Role of Epigenetics: Understand how epigenetics functions within the cell.
  • Definition: Recognize and define epigenetic modifications.

What is Epigenetics?

  • Function: Epigenetics allows cells to regulate:
    • Gene Expression: Determines which genes are expressed (i.e., when mRNA is made).
    • Timing of Expression: Controls when genes are turned on/off based on developmental state, external signals, and environmental responses.
    • Levels of Expression: Governs how much of a gene is expressed (none, a little, a lot).
  • Example: Viceroy butterfly (illustrating diverse phenotypes).

Definition and Characteristics of Epigenetics

  • Heritable Change: Epigenetic modifications represent heritable changes to the DNA sequence.
    • Mechanism: Heritability occurs through mitosis (cell division), not through meiosis (reproductive cell division).
    • Modification: Changes are modifications added to the DNA sequence without altering the sequence itself (As, Gs, Cs, Ts remain unchanged).

Importance of Epigenetic Control

  • Access Control: Epigenetic modifications influence whether DNA is accessible or not, which directly affects gene expression.

DNA Methylation

  • Outline Topics:
    • What DNA methylation is and its role in the cell.
    • General location in the genome.
    • Mechanism of copy through cell division.

Learning Objectives in DNA Methylation

  • Basic Structure: Understand the structural aspects of DNA methylation.
  • Inheritance Mechanism: Explain how DNA methylation patterns are transmitted during mitotic division.

Mechanism of DNA Methylation

  • Types: The focus is primarily on CpG methylation, which occurs when a cytosine (C) is methylated preceding a guanine (G) nucleotide.
  • Occurrence:
    • Methyl groups are added specifically to CpG dinucleotides.
    • It involves the addition of a methyl group to the cytosine base (C).
    • Key Enzymes:
    • DNMT3A and DNMT3B: De novo DNA methyltransferases that establish methylation patterns.
    • DNMT1: Maintenance DNA methyltransferase that propagates methylation patterns during cell division.
  • Example Sequence:
    • DNA Sequence: extATCGACTGCGAATTCext{ATCGACTGCGAATTC}
    • Methylation occurs at specific sites.

Epigenetic Inheritance

  • General Inheritance: DNA methylation patterns typically remain the same in daughter cells, which relates to epigenetic inheritance through mitotic processes rather than meiotic.
  • Situational Changes:
    • Changes observed in:
    • Gametogenesis and early development.
    • Tissue differentiation.
    • X chromosome inactivation.
    • Aging and imprinting.
    • Cancer pathology.

DNA Replication and Methylation

  • Process Details:
    • DNA replication results in hemi-methylation where one strand retains methylation while the new strand does not.
    • DNA polymerase does not recognize or copy methylation patterns; hence, DNMT1 reestablishes methylation post-replication.
    • Example sequences demonstrate this concept:
    • Methylation is indicated in both original and replicated strands.

Functional Role of DNA Methylation

  • CpG Methylation and Gene Expression:
    • Methylation at promoter regions signifies that the associated gene will not be expressed.
    • Reference to inactive X chromosomes and the gene promoters of other inactive genes.

Histones and Chromatin

  • Covered Topics:
    • Structure of chromatin, reasons for DNA wrapping, mechanisms by which cells modulate accessibility of DNA around histones.

Learning Objectives in Histones

  • Chromatin Structure: Grasp the structural aspects of chromatin.
  • Histone Modification Effects: Understanding how modifications (e.g., acetylation and methylation) affect the chromatin structure and consequently gene expression.

Chromatin Types

  • Closed Chromatin: Heterochromatin, referred to as "silent DNA".
  • Open Chromatin: Euchromatin, considered "active DNA", crucial for gene transcription.

Structure of Chromatin

  • Nucleosome: Basic unit of chromatin consists of:
    • Histone Proteins:
    • 2 x H2A, 2 x H2B, 2 x H3, 2 x H4.
    • DNA Length: Approximately 146 base pairs wrapped around histones
    • Linker DNA: Each nucleosome is separated by 20-60 bp, additional compaction facilitated by histone H1 binding.

Features of Histone Proteins

  • Charge Property: Histones are positively charged, facilitating attraction to negatively charged DNA.
  • Histone Tails:
    • Histones possess 'tails' rich in lysine residues that can undergo various modifications influencing chromatin structure dynamically.

Histone Tail Modifications

  • Modification Details:
    • Length: Tails average 20-40 amino acids long; lack defined secondary structure.
    • Functional Roles: Key in modulating the chromatin structure directly (neutralizing histone charges) or indirectly through recruitment of other proteins affecting chromatin accessibility.

Acetylation and Gene Expression

  • Acetylation:
    • Most prevalent modification of core histones.
    • Mechanism: Addition of an acetyl group neutralizes positive charges of histones allowing for increased DNA accessibility.
    • Result: Open chromatin structure, facilitating access for transcription factors and DNA polymerases, typically associated with active transcription.

Therapeutic Focus on Histone Modifications

  • HDAC Inhibition: Investigational drugs aim to inhibit histone deacetylases (HDACs) to promote gene expression.
    • Useful in conditions related to aberrant gene methylation, e.g., trichostatin A in breast cancer treatment.

Complexity of Histone Methylation

  • Mechanism: Involves the addition of one to three methyl groups to lysine residues.
    • Charge State: Methylation does not alter the charge of the histone tail.
    • Histone Mark Functionality: Different forms of lysine methylation (mono, di, tri) can indicate activation or repression of gene activity.

Histone Code and Gene Expression

  • Interpreting Modifications: Cells interpret combinations of histone marks, referred to as the "histone code", which informs overall gene expression patterns.
    • Unique Chromatin States: Over 50 distinct chromatin states associated with various biological functions have been identified.

Interaction of DNA and Histone Modifications

  • Chromatin State Effects:
    • Closed chromatin correlates with methylated DNA and deacetylated histones, resulting in gene transcriptional inactivity.
    • Open chromatin is characterized by non-methylated DNA and acetylated histones, leading to transcriptional activation.

DNA Methylation During Embryogenesis

  • Learning Objectives: Describe changes in DNA methylation patterns during gametogenesis and embryogenesis.
  • Developmental Changes: Methylation patterns are dynamic; influenced by both environmental and developmental factors during these critical processes.

Methods of Detecting DNA Methylation

  • Analytical Approaches:
    • Methylation-Sensitive Restriction Enzymes: Employed to distinguish between methylated and unmethylated DNA.
    • Bisulphite Modification: Converts unmethylated cytosines to uracils; methylated cytosines remain unchanged, allowing for differential PCR products.

Methylation-Sensitive Restriction Enzymes

  • Definition: These enzymes, a class of endonucleases, cut within specific DNA sequences, with characteristics dependent on their sensitivity to methylation.

  • Types:

    • Methylation-Insensitive Enzymes: Cut regardless of methylation status.
    • Methylation-Sensitive Enzymes: Will not cut if the DNA is methylated.

  • Example Enzymes:

    • Hpa II (methylation-sensitive)
    • Msp I (methylation-insensitive)

Analyzing Methylation Patterns

  • Methodology: Samples can undergo PCR or Southern blot analysis to differentiate methylation states.
    • Example presented (with references to genetic sequences and amplified products) helps demonstrate the concept of methylation analysis using restriction enzymes.

Bisulphite Treatment Technique

  • Chemical Procedure:
    • Cytosine Conversion: Bisulphite modifies cytosine bases into uracils which in turn pair with adenines during PCR.
    • PCR Outcomes: Unmethylated cytosines are converted to thymidines, while methylated cytosines remain unchanged, providing a means to assess methylation status in amplified DNA sequences.

Example of Bisulphite Sequencing

  • Sequence Outcomes: Showcases original and treated sequences to derive insights about methylation states based on preserved cytosines in PCR products.

Conclusion and Contact Information

  • Acknowledge Flinders University and the traditional custodians of the land where the University campuses are located.
  • Reference to Flinders University contact pages for further engagement that includes social media handles.