Epigenetics Study Guide

📖 Core Concepts Epigenetics – heritable (or semi‑stable) changes in gene expression without altering the DNA sequence. Epigenome – the full complement of epigenetic marks (DNA methylation, histone PTMs, non‑coding RNAs) in a cell. Chromatin – DNA wrapped around histone octamers; its packaging (euchromatin vs heterochromatin) dictates accessibility. DNA methylation – addition of a methyl group to the 5‑carbon of cytosine (5‑mC) in CpG dinucleotides, generally silencing promoters. Histone post‑translational modifications (PTMs) – chemical tags (acetyl, methyl, phosphate, etc.) on histone tails that remodel nucleosome‑DNA interaction. Non‑coding RNAs (ncRNAs) – RNAs that do not code for protein but guide or execute epigenetic regulation (miRNA, siRNA, lncRNA). Epigenetic inheritance – transmission of epigenetic states through mitosis (somatic) and/or meiosis (germ‑line). --- 📌 Must Remember DNA methyltransferases DNMT1 – maintenance methylation during replication (requires PCNA). DNMT3A/3B – de‑novo methylation in development. Acetylation → transcription – neutralizes lysine’s positive charge → looser DNA‑histone binding. Methylation of H3K9 & H3K27 → repressive heterochromatin (HP1 binding for H3K9me). H3K4me3 & H3K9ac → marks of active promoters. Promoter CpG methylation blocks transcription‑factor binding or recruits MeCP/MBD proteins → HDAC complexes → silencing. Ten‑Eleven Translocation (TET) enzymes oxidize 5‑mC → active demethylation. Imprinting – parent‑specific DNA methylation; loss on Chr 15 → Angelman (maternal loss) or Prader‑Willi (paternal loss). Bisulfite conversion – unmethylated C → U (read as T); methylated C remains C → base‑resolution methylation map. ChIP‑seq – maps protein‑DNA interactions or specific histone PTMs genome‑wide. --- 🔄 Key Processes DNA Methylation Maintenance Replication fork → DNMT1 recruited by PCNA → methylates hemimethylated CpG → restores symmetrical 5‑mC. De‑novo Methylation DNMT3A/3B + DNMT3L recognize unmethylated CpGs → add methyl groups (requires SAM as methyl donor). Active Demethylation (TET pathway) 5‑mC → 5‑hmC → 5‑fC → 5‑caC → base‑excision repair removes the oxidized base → unmodified C. Histone Acetylation Cycle HATs add acetyl groups → chromatin relaxation → transcription. HDACs remove acetyl groups → chromatin compaction → repression. Histone Methylation Cycle KMTs (SET‑domain) add 1‑3 methyl groups to lysine residues. KDMs (Jumonji C domain) demethylate via Fe(II)/α‑KG‑dependent oxidative reaction. ncRNA‑Directed Silencing siRNA → Argonaute complex → recruits DNMTs/HMTs to target promoter → DNA/histone methylation → transcriptional silencing. --- 🔍 Key Comparisons DNA methylation vs. Histone acetylation Methylation represses (especially promoter CpG); acetylation activates. DNMT1 vs. DNMT3A/3B DNMT1 = maintenance; DNMT3A/3B = de‑novo. H3K9me vs. H3K27me H3K9me → constitutive heterochromatin (HP1 binding). H3K27me → facultative heterochromatin (Polycomb repression). miRNA vs. siRNA miRNA: imperfect base‑pairing, mainly post‑transcriptional repression. siRNA: perfect pairing, can guide transcriptional silencing via DNA methylation. Bisulfite sequencing vs. Nanopore sequencing Bisulfite = chemical conversion, requires PCR; Nanopore = native DNA, detects methylation directly. --- ⚠️ Common Misunderstandings “All methylation silences genes.” Gene‑body methylation can correlate with active transcription; context matters. “Histone methylation is always repressive.” H3K4me3 is activating; the residue and methylation state (me1/me2/me3) dictate outcome. “Epigenetic changes are permanent.” Many marks are reversible (acetylation, TET‑mediated demethylation). “Only CpG islands are methylated.” Non‑CpG (CpA, CpT) methylation occurs in embryonic stem cells and neurons. --- 🧠 Mental Models / Intuition “Lock & Key” – DNA methylation = lock on promoter; HDAC complexes = key‑holder that keeps the lock closed. “Chromatin as a spring” – acetylation = loosening the spring (easy to pull apart → transcription); deacetylation = tightening. “Epigenetic code = traffic lights” – green lights (H3K4me3, H3K9ac) permit transcription; red lights (H3K9me3, H3K27me3) stop it. --- 🚩 Exceptions & Edge Cases CpG island promoters are usually unmethylated even in silent genes; repression relies on repressive histone marks. X‑chromosome inactivation uses both DNA methylation and H3K27me3; loss of one does not fully reactivate the chromosome. Bacterial adenine methylation (Dam) regulates replication timing, not the same as eukaryotic 5‑mC methylation. Histone lactylation – a metabolism‑linked PTM that can activate genes under high lactate conditions, not covered by classic acetyl/methyl rules. --- 📍 When to Use Which Diagnosing promoter silencing → Perform bisulfite PCR/sequencing for methylation status. Mapping active enhancers → Use ChIP‑seq for H3K27ac (activating) plus ATAC‑seq for accessibility. Identifying repressive domains → ChIP‑seq for H3K9me3 or H3K27me3. Assessing ncRNA impact → qRT‑PCR for specific miRNA/lncRNA levels; perform RIP‑seq to capture RNA‑protein complexes. Studying transgenerational effects → Combine gamete‑specific bisulfite sequencing with parent‑of‑origin allele‑specific expression assays. --- 👀 Patterns to Recognize Co‑occurrence of H3K4me3 + H3K27ac → active promoter/enhancer. H3K9me3 + DNA methylation at the same locus → stable heterochromatin (e.g., transposon silencing). Elevated TET expression + 5‑hmC peaks → regions undergoing active demethylation (often in neurons during learning). miRNA seed match + down‑regulated target mRNA → post‑transcriptional repression signature. CpG‑dense promoter + hyper‑methylation in tumor → classic tumor‑suppressor gene silencing pattern. --- 🗂️ Exam Traps “All histone methylation is repressive.” – Remember H3K4me3 is activating. “DNA methylation always occurs at CpG islands.” – Non‑CpG methylation exists, especially in brain. “Only DNMT1 is involved in cancer‑related methylation changes.” – DNMT3A/B over‑expression also drives de‑novo hyper‑methylation in tumors. “Bisulfite sequencing detects 5‑hmC.” – It cannot distinguish 5‑mC from 5‑hmC; specialized oxidative bisulfite or oxBS‑seq is required. “All epigenetic changes are inherited.” – Many are transient, responding to environmental cues; heritability must be demonstrated across cell divisions or generations. ---