Overview
This lecture covers epigenetics and epigenomics: how non-genetic factors (environment, lifestyle) modify gene expression without changing the DNA sequence. It works through the mechanics of DNA packaging into chromatin, how histone modifications and DNA methylation of promoters switch genes between active (euchromatin) and inactive (heterochromatin) states, how these marks are inherited and reset, how DNA methylation can itself cause mutation, and closes on the added complexity of gene networks.
Non-genetic influences on health and disease
- Health/disease outcomes depend on genes plus non-genetic inputs: environment and lifestyle choices.
- A cited breakdown of factors influencing disease: Lifestyle 53%, Environment 21%, Genetics/body makeup 16%, Medical 10%.
Epigenetics and epigenomics: definitions
- Epigenetics/epigenomics: chemical covalent modifications of DNA (of individual genes, or of the whole genome) that change gene/genome regulation without altering the DNA sequence.
- Epigenetics: effect on gene expression from chemical modification of a single gene.
- Epigenomics: effect on gene expression from chemical modification of the whole genome (many genes at once).
- These modifications are determined by changing cellular needs, external environment, and lifestyle choices.
- They can be dynamic (change in response to stimuli) or stable (passed onto future generations).
- Every individual is biochemically unique: gene products reflect the combination of genome sequence plus each person’s lifetime pattern of environmental exposures and lifestyle choices, which chemically modify DNA and proteins and thereby modify gene expression.
DNA packaging into chromatin
- Chromatin = DNA + histones; different degrees of DNA packaging give different chromatin forms.
- Packaging hierarchy: DNA double helix (2 nm) -> wrapped around histones forming nucleosomes (“beads on a string”, 10 nm fiber) -> 30 nm fiber -> looped domains (300 nm fiber) -> replicated chromosome (1400 nm) -> metaphase chromatid (700 nm). This packages DNA about 10,000-fold shorter than its extended length.
- Chromatin is dynamic and moves between two forms:
- Euchromatin: loosely packed (10 nm fiber).
- Heterochromatin: densely packed (30 nm fiber).
- Nucleosome structure: a core cluster of 8 histone proteins (two each of H2A, H2B, H3, H4), with DNA wrapped around it twice. Each histone has a tail rich in positively charged amino acids.
- The positively charged histone tails bind the negatively charged DNA backbone; this binding is what converts loose 10 nm chromatin into dense 30 nm chromatin.
- Regulatory consequence:
- Heterochromatin -> inactive gene: transcription factors and RNA polymerase II cannot access the gene.
- Euchromatin -> active gene: transcription factors and RNA polymerase II can access the gene.
- Key summary given in the lecture: DNA is condensed by histones binding it via their positively charged tails. Condensed (heterochromatin) regions have inaccessible promoters -> no transcription. Uncondensed (euchromatin) regions have accessible promoters -> transcription proceeds.
Mechanisms that switch chromatin between hetero- and euchromatin
Four cooperating (non-mutually-exclusive) mechanisms:
- Posttranslational covalent chemical modification of histone tail amino acids: acetylation, methylation, phosphorylation, ubiquitylation, sumoylation. These alter chromatin structure directly or recruit histone modifiers.
- Histone variants with distinct properties that create localised hetero- or euchromatin regions.
- Histone chaperones that regulate the supply of histone proteins.
- ATP-dependent complexes that physically slide nucleosomes along chromatin or remove histones.
One-letter codes used: K = Lysine (Lys, positive), R = Arginine (Arg, positive), S = Serine (Ser).
Histone acetylation and methylation
- Acetylation: addition of an acetyl group (CH3CO) to lysine. Neutralises the lysine’s positive charge, decreasing histone tail-DNA interaction, so condensed 30 nm chromatin relaxes into 10 nm chromatin. Makes genes accessible for transcription (-> euchromatin).
- Demethylation (removal of methyl groups from lysine/arginine): usually makes genes accessible for transcription. Methylation does not change the charge of the residue, but hydrophobic methyl groups attract each other, so methylated nucleosomes pack tightly, blocking gene access.
- Methylation (addition of methyl groups): usually condenses chromatin further (-> heterochromatin), because the hydrophobic methyl groups attract one another.
- Methylation can decrease (usually) or increase (uncommon) transcription depending on which residues are methylated and how many methyl groups are attached.
- Summary of opposite effects:
- Acetylation -> euchromatin (active/transcribed genes).
- Deacetylation -> heterochromatin (inactive genes).
- Methylation -> heterochromatin (inactive genes), usually.
- Demethylation -> euchromatin (active genes), usually.
- The combination of modification type, site and degree determines the net effect on chromatin remodeling and transcription.
- Histone modifications act:
- Directly, by remodeling chromatin structure to control gene accessibility.
- Indirectly, by recruiting other proteins that activate or inactivate transcription (e.g. via chromatin remodeling or bringing in transcription-activating/inhibiting proteins).
Take-home for slides 65-77: chemical modification of histone tails (methylation, acetylation) shifts DNA between heterochromatin (promoters inaccessible, no transcription) and euchromatin (promoters accessible, transcription occurs); environmental and lifestyle factors can modulate these histone modifications.
DNA methylation of gene promoters
- DNA methylation: addition of a methyl group to the base cytosine (C), producing 5-methylcytosine (5-mC). When this occurs in a gene’s promoter region, it inactivates that gene’s expression.
- Unmethylated promoters allow transcription into the downstream gene; methylated CpG sites in/near the promoter block transcription.
- Mechanism of inhibition: methyl groups are recognised and bound by methyl-binding proteins (MBPs), which either:
- physically block transcription factor and RNA polymerase II access to the promoter, or
- recruit enzymes that deacetylate histones in that region, remodeling the chromatin into heterochromatin.
- The cytosine that gets methylated is the C of a CpG (cytosine-guanine) dinucleotide.
- Highly/constantly expressed genes (“housekeeping genes”) have unmethylated CpG islands in their promoters.
- Inactive/rarely expressed genes have methylated CpG islands in their promoters.
- Note: a promoter only contains a TATA box if its gene needs to be switched on and off frequently under varying conditions.
- Inheritance of methylation pattern: after DNA replication, each daughter duplex is hemimethylated (one parent strand methylated, one new strand not); a subsequent methylation step restores full methylation on both daughter duplexes, preserving the parental pattern. This lets each cell type maintain its own methylation pattern and thus its own characteristic protein expression.
- Epigenetic marks (including methylation) are normally erased in the germ line, resetting the embryo’s gene expression to the original start state.
- Contrast with genetic inheritance: a DNA sequence change that silences a gene is passed unchanged through the germ line to all descendants.
- Epigenetic inheritance: a chemical modification that silences a gene is normally reset during germ cell production, so the gene can return to “on” in the next generation.
- DNA methylation and other epigenetic modifications regulate gene expression during normal development; dysregulation of these leads to abnormal gene expression and disease. Global 5-methylcytosine levels fluctuate substantially across developmental stages (oocyte/sperm -> zygote -> ICM -> implantation -> fetal brain/germ cell development -> child -> adult), with a dip around implantation, and different steady-state levels reached in different lineages by adulthood.
Take-home for slides 79-90: promoter methylation decreases or abolishes transcription of that gene; methylation patterns can be modulated by environmental factors (e.g. smoking, toxins, viruses, medication, diet, exercise, sleep, alcohol, stress) and can be transmitted through generations; DNA methylation can also directly cause a C -> T mutation; gene networks make regulation of gene expression very complex.
DNA methylation can cause DNA sequence change
- Methylated cytosine is prone to spontaneous deamination, converting it to thymine: Cytosine -> (methylation) -> 5-methylcytosine -> (deamination) -> Thymine.
- Increased methylation driven by environmental stimuli or lifestyle factors can therefore increase the rate of cytosine-to-thymine point mutations.
- Other chemical deamination reactions change base-pairing properties and can also cause mutation:
- Adenine -> hypoxanthine (HX)
- Guanine -> xanthine (X)
- Cytosine -> uracil (U, in DNA)
- X-ray/UV radiation can chemically modify thymine so it pairs with guanine instead.
- Chemicals in antibacterial/antifungal cleaning agents can form ethylguanine (eG), which pairs with thymine instead of cytosine, and can generate a mutant sequence (illustrated: -ATTGA-/-TAACT- becoming a G-to-T-derived mutant sequence via an eG:C mismatch).
Gene networks
- Gene expression at the right time, place and amount is guided by the combination of epigenetic/epigenomic marks (histone modifications, DNA methylation) and the DNA sequence itself.
- Almost all genes function as part of gene networks: a single gene can enhance or decrease the activity of many other genes.
- Gene networks are themselves influenced by environmental and lifestyle factors acting through epigenetics/epigenomics.
- This network structure makes regulation of gene expression very complex (illustrated with a large network of interacting genes/proteins, e.g. collagen genes, MMP2, FN1, ELN, SPARC).
Self-test
- Define epigenetics and distinguish it from epigenomics.
- Describe the steps by which histone tails convert loosely packed (10 nm) chromatin into densely packed (30 nm) chromatin, and explain why this affects transcription.
- List the four mechanisms by which cells switch between heterochromatin and euchromatin.
- Distinguish the effect of histone acetylation from histone methylation on chromatin structure and gene expression, including the underlying chemical reason for each.
- Explain the two mechanisms by which methyl-binding proteins (MBPs) silence a methylated gene promoter.
- Distinguish genes with unmethylated CpG islands from genes with methylated CpG islands in terms of expression level, and give an example gene category for each.
- Describe how DNA methylation patterns are maintained through cell division, and explain why this allows different cell types to express different proteins.
- Distinguish epigenetic inheritance from genetic inheritance in terms of what happens to the silencing signal during germ cell production.
- Describe the chemical steps by which DNA methylation can lead to a permanent point mutation.
- Explain why gene networks make it difficult to predict the overall effect of a single epigenetic change on gene expression.
Answers
Reveal answers
- Epigenetics is the study of chemical covalent modifications to an individual gene that alter its regulation without changing its DNA sequence; epigenomics is the same concept applied across the whole genome, affecting many genes/proteins at once rather than a single gene.
- Histone tails carry many positively charged amino acids; these tails bind the negatively charged DNA backbone. This tail-DNA binding pulls the DNA into a more tightly packed conformation, converting the loosely packed 10 nm (euchromatin) fiber into the densely packed 30 nm (heterochromatin) fiber. Because heterochromatin is inaccessible to transcription factors and RNA polymerase II, this reduces or blocks transcription of genes in that region.
- (1) Posttranslational covalent chemical modification of histone tail amino acids (acetylation, methylation, phosphorylation, ubiquitylation, sumoylation); (2) use of histone variants with distinct properties; (3) histone chaperones regulating histone supply; (4) ATP-dependent complexes that slide or remove nucleosomes.
- Acetylation adds an acetyl group to lysine, neutralising its positive charge; this weakens the histone tail’s grip on DNA, relaxing chromatin into euchromatin and activating transcription. Methylation adds methyl groups without changing charge, but the hydrophobic methyl groups attract each other, packing nucleosomes more tightly into heterochromatin and usually repressing transcription (though the direction depends on which residue is methylated).
- Bound MBPs either (a) physically block transcription factors and RNA polymerase II from accessing the promoter, or (b) recruit enzymes that deacetylate the local histones, remodeling the chromatin into heterochromatin.
- Genes with unmethylated CpG islands in their promoters are highly/constantly expressed (“housekeeping” genes); genes with methylated CpG islands are inactive or expressed only rarely/intermittently.
- After DNA replication each daughter duplex is only hemimethylated (methyl marks on the old strand only); a maintenance methylation step then re-methylates the new strand, restoring full methylation identical to the parent. This lets each cell type propagate its own stable methylation pattern to daughter cells, so it keeps expressing the protein set specific to that cell type.
- In genetic inheritance, a DNA sequence change that silences a gene is passed on permanently and unchanged through the germ line. In epigenetic inheritance, a chemical modification silencing a gene is normally erased/reset during germ cell production, so the gene returns to its original “on” state in the next generation.
- Methylation converts cytosine to 5-methylcytosine; 5-methylcytosine is prone to spontaneous deamination, which converts it into thymine, producing a permanent C-to-T point mutation in the DNA sequence.
- Because almost every gene is embedded in a network where it can enhance or suppress many other genes, an epigenetic change to one gene can propagate through the network to affect the expression of numerous other genes, so its net effect on the system cannot be predicted from that one gene in isolation.