BIO 412 Lecture 12: Epigenetics and Chromatin Remodeling Study Guide

Defining Epigenetics and Heritability

• Epigenetics Definition: Literally translates to "changes on top of genetics." It refers to gene expression changes that occur without direct alterations to the actual DNA sequence.
• Operational Definition: An epigenetic trait is defined as a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.
• Heritability: Epigenetic phenotypes are heritable, meaning they are passed on through either mitosis (cell division) or meiosis (reproduction).
• CDC Definition: According to the Centers for Disease Control and Prevention (CDC), epigenetics is the study of how behaviors and environment can cause changes that affect the way your genes work.

Hierarchical Levels of Eukaryotic DNA Compaction

• DNA Strand: The basic unit of genetic material, measuring $2\,nm$ in diameter.
• Nucleosome ("Beads on a String"): DNA wraps around a nucleosome made of histones. This structure forms a long strand with a diameter of $11\,nm$.
• Chromatin Fiber: The "beads on a string" coil into a helical structure, producing a chromatin fiber with a diameter of $30\,nm$.
• Further Condensation: The chromatin fiber condenses further into loops, scaffolds, and domains, reaching a diameter of $700\,nm$.
• Metaphase Chromosome: In its most condensed form, a duplicated metaphase chromosome is $1400\,nm$ in diameter and approximately $2\,\mu m$ in length.

Primary Epigenetic Mechanisms and Indicators

• Influencing Factors: Epigenetic mechanisms are affected by development (in utero and childhood), environmental chemicals, drugs/pharmaceuticals, aging, and diet.
• DNA Methylation:
  • Methyl groups (epigenetic factors found in some dietary sources) tag DNA.
  • This tagging can activate or repress genes.
  • When DNA is methylated, it often becomes inaccessible, rendering the gene inactive.
• Histone Modification:
  • Histones are proteins around which DNA winds for compaction and gene regulation.
  • Epigenetic factors bind to histone "tails," altering the extent to which DNA is wrapped.
  • This modification determines the availability of genes in the DNA to be activated; accessible DNA allows for active genes.
• Health Endpoints: Epigenetic changes are linked to various health outcomes, including cancer, autoimmune diseases, mental disorders, and diabetes.

Structural Composition of Nucleosomes and Histones

• Chromatin: The complex of DNA and packaging proteins that make up chromosomes. Packaging proteins help condense DNA into a small space and protect the genome.
• Basic Packaging in Bacteria: Small positively charged proteins bind along the DNA to counteract the negative charges on the DNA phosphate backbone, bending and compacting it into a structure called the nucleoid.
• Eukaryotic Binding Proteins: In eukaryotes, these basic proteins are called histones. There are four core histones: $H2A$, $H2B$, $H3$, and $H4$.
• Conservation: These core histones arose early in evolution and are very highly conserved, as they are crucial for survival.
• Charge Dynamics: Histones are rich in basic amino acids such as lysine and arginine, which carry positive charges and stabilize the DNA-histone interaction.
• The Histone Octamer: The center of the nucleosome is an octamer consisting of two of each core histone ($2 \times H2A$, $2 \times H2B$, $1 \times H3$, and $1 \times H4$).
• Nucleosome Dimensions: A nucleosome consists of a histone octamer plus approximately $146\,bp$ of duplex DNA. The linker DNA between nucleosomes is roughly $20-90\,bp$.

Nucleosome Assembly and DNA Interaction

• Assembly Process:
  1. The $H3-H4$ heterotetramer associates with the DNA.
  2. Two $H2A-H2B$ dimers then associate to complete the formation of the octamer.
• Left-Handed Wrapping: DNA winds around the histone octamer in a left-handed direction. This specific winding means that when the octamer is removed, the remaining DNA is negatively supercoiled.
• Facilitation of Cell Processes: Negative supercoiling makes strand separation easier, which is essential for the processes of replication and transcription.

Histone Tails and Intrinsically Disordered Regions (IDRs)

• N-Terminal Tails: Each core histone possesses an N-terminal "tail" that extends outwards between the coils of DNA.
• Tail Characteristics: These tails consist of up to $25$ amino acids and have an undefined structure. They are described as intrinsically disordered (containing IDRs).
• Flexibility and Function: Because they are flexible and dynamic, histone tails can respond to evolving gene expression needs. They interact with other nucleosomes to aid in further DNA compaction.
• Chemical Modifications: These tails are the primary sites for chemical modifications that define chromatin structure and function.

Histone Variants and Special Chromatin Locations

• Variant Incorporation: While the four core histones are most common, other variants can be incorporated into nucleosomes at specific chromatin locations.
• $H2A.X$ Example: This histone variant is phosphorylated at sites where DNA double-strand breaks occur. Once phosphorylated, it recruits repair machinery to the damaged site.

Euchromatin vs. Heterochromatin: Dynamic States of Compaction

• Differential Compaction: Chromosomes exhibit varying degrees of compaction throughout the cell cycle. During interphase, chromosomes are relatively uncondensed, but they show a range of compaction along their length.
• Euchromatin:
  • Relatively decondensed, open, and active regions.
  • Stains lightly.
  • Associated with high levels of transcription.
• Heterochromatin:
  • More compacted, closed, and inactive regions.
  • Stains darkly.
  • Usually resists transcription (though some does occur).
  • A gene translocated from euchromatin to heterochromatin can be actively prevented from being transcribed.

Categories of Heterochromatin: Constitutive and Facultative

• Regional Dominance: Some chromosome regions are rich in heterochromatin, specifically telomeres (the ends), centromeres, and regions with highly repetitive DNA sequences.
• Constitutive Heterochromatin:
  • Typically contains very few genes.
  • Consists primarily of highly repetitive sequences.
  • Always condensed and inactive in all cell types.
• Facultative Heterochromatin:
  • Contains genes that are silenced in a cell-type specific manner (e.g., developmental genes active only in specific cell types).
  • Can be dynamically regulated to alternate between active and inactive states based on environmental cues or cell type.
• Properties of Heterochromatin: It is more resistant to nuclease digestion than euchromatin. Low recombination rates in heterochromatic regions help protect sensitive parts of the genome, such as ribosomal RNA synthesis genes.

Post-Translational Modifications (PTMs) of Histones: Overview

• Dynamic Regulation: Side chains of histones undergo modifications that act as signals for gene regulation. These modifications are reversible.
• Major PTM Types:
  • Acetylation.
  • Methylation.
  • Phosphorylation.
  • Ubiquitination.
• Signals and Recruitment: Modifications recruit specific proteins to the chromatin. Specialized enzymes add or remove chemical groups based on the metabolic demands of the cell.

Snapshot of Chemical Histone Modifications

• Methylation (K, R): Represented by Me.
• Acetylation (K, S, T): Represented by Ac.
• Propionylation (K): Represented by Pr.
• Butyrylation (K): Represented by Bu.
• Crotonylation (K): Represented by Cr.
• 2-Hydroxyisobutyrylation (K): Represented by Hib.
• Malonylation (K): Represented by Ma.
• Succinylation (K): Represented by Su.
• Formylation (K): Represented by Fo.
• Ubiquitination (K): Represented by Ub.
• Citrullination (R): Represented by Cit.
• Phosphorylation (S, T, Y, H): Represented by Ph.
• Hydroxylation (Y): Represented by HPh.
• O-GlcNAcylation (S, T): Represented by Og.
• ADP Ribosylation (K, E): Represented by Ar.

Histone Acetylation: Mechanics and Enzymes

• Widespread Occurrence: Most eukaryotic chromosome regions contain histones with acetylated lysines. Euchromatin possesses more acetylation than heterochromatin.
• Enzymatic Activity:
  • Histone Acetyltransferases (HATs): Add acetyl groups to lysine side chains.
  • Histone Deacetylases (HDACs): Remove acetyl groups.
• Transcriptional Impact: Acetylation removes the positive charge from lysine, weakening the interaction between histones and the negatively charged DNA. This creates an open conformation associated with active transcription.
• Bromodomains: Acetylated lysines generate binding sites for bromodomain proteins. These proteins then recruit nucleosome remodeling complexes.

Histone Methylation: Regulation of Gene States

• Methyl Capacity: Up to three methyl groups can be added to a lysine residue, and up to two can be added to an arginine residue.
• Enzymatic Activity:
  • Histone Methyltransferases (HMTs): Add methyl groups.
  • Histone Demethylases: Remove methyl groups.
• Position matters: The specific effect (activation or repression) depends on the exact residue modified.
  • H3 Lysine 9 (H3K9): Methylation is associated with silent chromatin (heterochromatin).
  • H3 Lysine 4 (H3K4): Methylation is associated with active chromatin.
• Chromodomains: These bind to specific methylated lysines and are often associated with transcriptional silencing.

Histone Phosphorylation and the Histone Code Hypothesis

• Regulatory Signals: Phosphates are added by kinases and removed by phosphatases.
• Functional Examples:
  • H3 Serine 10 (H3S10): Phosphorylation allows cell growth transcription to occur.
  • Mitosis: Phosphorylation of H3 serine 10 and serine 28 correlates with chromosome condensation during mitosis.
• The Histone Code: This hypothesis suggests that unique combinations of modifications define specific chromatin states and dictate gene expression. Modifications can influence one another:
  • Phosphorylation at serine 10 promotes acetylation of lysine 14.
  • Acetylation of lysine 14 inhibits methylation of lysine 9.

Histone Ubiquitylation and Sumoylation

• Ubiquitin: Unlike smaller chemical groups, ubiquitin is a large, $76$-amino acid protein. It is added via the E1-E2-E3 enzymatic system and removed by deubiquitinating enzymes (DUBs).
• Effect on Expression: Ubiquitylation on $H2A$ or $H2B$ can either suppress or enhance gene expression depending on the specific attachment site.
• Proteolysis Target: Ubiquitin can target histones for destruction. For example, the $CENP-A$ histone variant is polyubiquitinated and targeted for proteolysis to prevent it from being incorporated into non-centromeric DNA.
• Sumo (Small Ubiquitin-like Modifier): Sumoylation facilitates gene silencing by recruiting histone deacetylases and heterochromatin proteins.

Summary of Functional Outcomes for Histone Tail PTMs

• $H1$ Phosphorylation: Chromatin condensation; gene-specific activation and repression.
• $H2A$ Acetylation: Transcriptional activation; essential for Tetrahymena survival.
• $H2A$ Ubiquitination: Transcriptional repression.
• $H2B$ Ubiquitination: Prerequisite for $H3$ methylation.
• $H3$ Acetylation: Chromatin remodeling and transcriptional activation.
• $H3$ Methylation (K4, R17): Transcriptional activation.
• $H3$ Methylation (K9, K79): Transcriptional repression.
• $H4$ Acetylation: Nucleosome loosening and transcriptional activation.
• $H4$ Methylation (K20): Transcriptional repression.

Principles and Mechanisms of Chromatin Remodeling

• The Barrier Problem: Chromatin packing acts as a physical barrier to proteins needed for replication and transcription.
• ATP-Dependent Complexes: These use energy from ATP to increase DNA accessibility via three primary mechanisms:
  1. Sliding: Moving the histone octamer along the DNA.
  2. Removal: Taking the histone octamer off DNA and transferring it elsewhere.
  3. Looping: Introducing transient loops into the DNA.

Classes of Nucleosome Remodeling Complexes and Subunits

• SWI/SNF: Disrupts standard nucleosome positioning.
• ACF (ISWI family): Functions to position nucleosomes during chromatin assembly.
• Structural Subunits:
  • ATPase domain: Found in all complexes; composed of two parts: Dexx and HELICc.
  • Bromodomain: Binds to acetylated lysines.
  • Chromodomain: Binds to methylated lysines.
  • SANT-SLIDE: Binds to histone tails.
• Recruitment: Remodeling complexes are precisely recruited by transcription activator proteins (sequence-specific binding) or following specific histone modification patterns.

Epigenetic Modulators in Cancer Therapy: HDAC Inhibitors

• Medical Utility: Histone Deacetylase inhibitors (HDACis) are used to treat malignancies such as leukemia, B-cell lymphoma, multiple myeloma, and virus-associated tumors.
• FDA Approved Inhibitors:
  • Vorinostat: Used for persistent or recurrent cutaneous T-cell lymphoma.
  • Belinostat: Used for relapsed or refractory peripheral T-cell lymphoma.
  • Romidepsin: Used for cutaneous T-cell lymphoma.
  • Panobinostat: Used for relapsed multiple myeloma.
• Limitations: HDAC inhibitors have shown limited success in the treatment of solid tumors.

Mitochondrial Nucleoids and mtDNA Organization

• Nucleoid Structure: Mitochondrial DNA ($mtDNA$) is not organized into histones; instead, it forms structures called nucleoids. These consist of DNA-binding core proteins involved in maintenance and transcription.
• Peripheral Factors: These signals control mitochondrial biogenesis, apoptosis, metabolism, and retrograde signaling (mitochondria-to-nucleus).
• $TFAM$: The primary transcription factor and $mtDNA$-binding protein that works analogously to histone proteins in the mitochondria.

DNA Methylation: CpG Islands and Promoter Silencing

• CpG Islands (CGIs): Short stretches of palindromic DNA defined as "Cytosine-phosphodiester linkage-Guanine." They code for the same sequence ($5' \rightarrow 3'$) on both complementary strands.
• Distribution: CGIs extend between $300-3000\,bp$ and are associated with approximately $40\%$ of mammalian gene promoters.
• Gene Silencing: These regions regulate expression through transcriptional silencing. This is critical for processes like X-inactivation, genomic imprinting, and cell differentiation.

CpG Islands in Cancer: Hypermethylation and Tumor Suppression

• Normal State: CpG islands typically lack DNA methylation in healthy cells.
• Aberrant Hypermethylation: In cancer, CGIs in promoter regions often become hypermethylated. This silences tumor suppressor genes, driving tumor progression and developing the "Hallmarks of Cancer."

Microsatellite Instability (MSI) and Colorectal Cancer

• Microsatellites: Short tandem repeats of DNA that are highly prone to replication errors due to their repetitive nature.
• $MLH1$ Methylation: Methylation of the $MLH1$ promoter is common in sporadic microsatellite unstable tumors (colorectal and endometrial cancer). This leads to the loss of $MLH1$ protein expression.
• MSI Phenotype: Without $MLH1$ (a mismatch repair protein), cells accumulate insertion/deletion (indel) mutations. Roughly $80-90\%$ of sporadic MSI colorectal cancers (CRCs) are caused by $MLH1$ promoter methylation.

Molecular Mechanism of DNA Polymerase Slippage

• Process of MSI:
  1. The polymerase synthesizes a repeated sequence.
  2. The polymerase reaches a barrier (such as a hairpin structure) on the lagging strand and pauses.
  3. Polymerase dissociation occurs.
  4. If the barrier is not disrupted, the tip of the newly synthesized strand unpairs from the template and anneals to the second repeat beyond the barrier. Synthesis then resumes.
• Consequences:
  • Loop out of new strand: Results in the addition (insertion) of an extra nucleotide base.
  • Loop out of template strand: Results in the omission (deletion) of a nucleotide base.

Revising the Central Dogma: The $P = F^3E$ Model

• The Genotype-Phenotype Equation: The original module equation is revised from Phenotype = Form, Function, Factors ($P = F^3$) to Phenotype = Form, Function, Factors in the environment, AND Epigenetics ($P = F^3E$).
• Refined Rule: Genetic material (DNA/RNA) is subject to mutation and epigenetic changes, which together serve as the targets for evolution.
• The Role of RNA: RNA links DNA (information) with proteins (cellular actions). All RNAs are involved in translation, and non-transcriptional/non-translational mechanisms for protein synthesis exist.
• Protein Autocatalysis: Proteins undergo post-translational modifications and folding to become functional, and some exhibit autocatalytic modification capabilities.

Course Administrative Details and Final Exam Information

• Quiz #10: accessible on Brightspace at 5:55 PM EST. Duration: 20 minutes. Format: 5 multiple choice questions (2 points each; 10 points total). Password: QUIZ#10izdiEnD!.
• Course Evaluation: Required 100% participation; due by 5/19/26 at 11:59 PM.
• Final Exam Schedule: Date is May 18th, 2025. Time: 6-00 PM to 8-00 PM EST. Lateness over 10 minutes results in disqualification from the exam.
• Exam Content: Coverage includes Lectures 9 through 12, with emphasis on mastering foundational knowledge from Lectures 1 through 8. The format includes 5 short response questions.
• Grades: Final grades are submitted to the University by 5/29/26. The instructor will have grades ready by 5/27 or 5/28 for discrepancy discussions.

Questions & Discussion

• Question: Why does DNA need to be packaged in the nucleus by proteins?
• Question: Does chromatin exist in one isolated state or is it highly dynamic in structure and chemical composition?
• Question: What type of genes would always need to be repressed? What genes would need to be conditionally expressed? Why?
• Question: What function does chromatin remodeling serve in eukaryotic cells?
• Question: How does everything learned today impact the original definition of the central dogma of molecular biology?