Genome Structure and Regulation Lecture Notes on Regulation

General Genome Architecture and Eukaryotic Diversity

  • Conceptual Foundation: As Bill Bryson states in "A Short History of Nearly Everything," "all life is one," representing a profound biological truth regarding shared genetic foundations.
  • Physical Constraints of the Human Genome:
    • Length: The human genome spans approximately 2 meters2\,meters in length.
    • Compaction Requirements: It must be condensed to prevent tangling and degradation and to facilitate transmission to daughter cells.
    • Organization: Genomes are wound around nucleosomes and condensed into chromosomes within the cell nucleus.
    • Mitotic State: During mitosis, chromosomes consist of two identical strands known as sister chromatids.
    • Chromatin: This is unraveled, accessible material composed of DNA and proteins, including histones and proteins involved in transcription, replication, repair, recombination, and topology. The overall architecture of the chromosome serves as a regulator for gene expression.
  • Viruses and Prokaryotes:
    • Viral Genomes: Can be ssRNA, dsRNA, or dsDNA. The smallest is the Hepatitis Delta Virus at 1700 nt1700\,nt.
    • Bacterial Genomes: Typically circular (rarely linear) and millions of base pairs long.
    • Compaction: A 1 mm1\,mm bacterial chromosome must be condensed into a 1 μm1\,\mu m space using histone-like proteins.
    • Configuration: Usually one copy of the chromosome exists in the nucleoid region, often accompanied by additional plasmids.
  • Eukaryotic Genomes:
    • Size: Millions to billions of base pairs bundled into 2 to ~100 chromosomes (humans possess 46).
    • Ploidy: Typically diploid with 2 homologs.
    • Extreme Examples:
      • Ciliates: Over 15,00015,000 chromosomes with 19001900 copies of each.
      • Male Jack Jumper Ants: 1 chromosome.
      • Butterflies: Up to 452 chromosomes.
    • Haploidy: Found in cnidarians, algae, plants, fungi, and some insects.
    • Polyploidy: Allows for increased RNA and protein generation. Megakaryocytes (for platelet production) have 64 copies of each chromosome. A study of heart cells found they were 85%85\% diploid and 15%15\% tetraploid at age two; by age twelve, they were equal; in adults, they were 27%27\% diploid, 71%71\% tetraploid, and 2%2\% octaploid.

Comparative Genomics: Humans, Primates, and Ancient Hominins

  • Complexity and Gene Number:
    • Complexity is more closely linked to gene regulation and organization than the sheer number of genes. For example, a fruit fly (DrosophilaDrosophila) has 13,60113,601 genes—50005000 fewer than a nematode worm—yet exhibits complex behaviors like mating, aging, and sensory perception.
    • Richard Dawkins notes that humans and worms share similar protein repertoires; the difference lies in the complicated sequences and spatial ranges in which these proteins are "called into action."
  • Ancestral Lineage:
    • Common Mammalian Ancestor: Lived 180 million180\,million years ago with 40 chromosomes and 12151215 conserved blocks of genes.
    • Last Universal Common Ancestor (LUCA): 355355 genes traced back to LUCA, which was anaerobic, CO2CO_2-fixing, H2H_2-dependent (Wood–Ljungdahl pathway), N2N_2-fixing, and thermophilic. Modern relatives include Clostridia and methanogens found in deep-sea vents.
  • Chromosome 2 Fusion:
    • Chimpanzees, bonobos, gorillas, and orangutans have 24 chromosomes. Humans, Neanderthals, and Denisovans have 23.
    • Human Chromosome 2 resulted from a fusion of two primate homologs, evidenced by a vestigial centromere and telomeres.
    • This fusion added 15,00015,000 new base pairs, possibly from Chromosome 9, involving transcription control and growth.
  • Atypical Human Ploidy: The "44 Chromosome Man" has a fusion of Chromosomes 14 and 15. He is phenotypically normal but has a family history of miscarriages. His offspring with a 46-chromosome female would carry one copy of the fusion.
  • Neanderthals and Denisovans:
    • Humans and Neanderthals are 99.84%99.84\% genetically identical, differing by fewer than 100 proteins.
    • Modern humans (especially non-Africans) carry 2−6%2-6\% Neanderthal DNA. Collective survival of the Neanderthal genome in humans is ~40%40\%.
    • Specific Neanderthal genes: 152 genes involved in fighting RNA viruses; progesterone receptor variant carried by 1 in 3 European women (linked to increased fertility and fewer miscarriages).
    • Oceania populations carry up to 8%8\% Denisovan DNA.
  • Human-Chimpanzee Divergence:
    • DNA Homology: 98.77%98.77\% identical at the sequence level (~1.2%1.2\% difference or 35 million35\,million SNPs). When inversions/indels are considered, it drops to ~96%96\%.
    • Changes: Humans gained ~689 genes and lost ~86 genes since the split. Y chromosomes show the most divergence.
    • Protein Homology: 99.1%99.1\% identical; 2/3rds2/3rds of proteins are 100% identical. Differences average only two amino acids.
    • Expression differences: Up to 40%40\% difference in protein expression levels; 90%90\% of genes are up-regulated in the human brain compared to chimps.
  • Specific Genetic Differences:
    • FOXP2FOXP2: Involved in reading/writing; differs by 2 of 715 amino acids.
    • ARHGAP11BARHGAP11B: Causes larger brain stem cell pools and neocortex folding; absent in chimps.
    • DUF1220DUF1220: Linked to higher cognitive function; humans have 212 copies, primates 37, mice 1.
    • MYH16MYH16: Lost in humans (myosin variant in jaw), allowing for smaller jaw muscles and enabling brain expansion.
    • AmylaseAmylase: Humans have 3×3\times more copies for starch digestion.

Genes, Intergenic DNA, and Repetitive Elements

  • Genome Density:
    • Viruses: Very dense; use both strands and overlapping genes.
    • E.coliE. coli: Almost entirely genes (46234623). 1600 have unknown function; 220 are pseudogenes.
    • Humans: Low gene density due to intergenic sequences and introns. Only 1.5%1.5\% is protein-coding.
  • Overlapping Genes:
    • Prokaryotes: Usually on the same strand; 1/3rd1/3rd overlap by ~2 nt2\,nt (mostly +2 frame).
    • Humans: Over 12001200 overlapping genes, typically antiparallel. Mitochondrial DNA (mtDNAmtDNA) contains 37 genes and significant overlaps (e.g., MT−ATP8MT-ATP8 and MT−ATP6MT-ATP6).
  • Intergenic sequences ( 25%~25\% of the genome):
    • Fragments of genes, regulatory sequences, or sequences of unknown function.
    • 8%8\% consists of nearly complete ancient viral genomes.
    • Pseudogenes: Nonfunctional mutant genes; ~13,00013,000 in humans. Can result from DNA polymerase slippage, unequal crossing over, or retrotransposition. Some can be reactivated (e.g., retrocyclin for HIV immunity).
  • Repetitive DNA (50−67%50-67\% of the genome):
    • Microsatellites: 5-50 units of ≤13 bp\le 13\,bp tandem repeats (e.g., TATATATA). Used in forensics.
    • Transposable Elements:
      • LINEs: Long Interspersed Nuclear Elements (LINE−1LINE-1). Active in humans but more so in chimps (10×10\times).
      • Alu Elements: Retrotransposons descended from signal recognition particles. 300 bp300\,bp long, 1.5 million1.5\,million copies (11%11\% of the genome). Involved in the loss of GULO (Vitamin C synthesis) and BRCA1 mutations.
  • Endogenous Retroviruses (ERVs): 5-8% of the genome. Derived from ancient infections. Essential for placenta development (syncytinsyncytin) and pancreatitis amylase expression.

RNA Transcriptome and Regulation

  • mRNA Structure:
    • 5' UTR: 7-methyl-G cap; regulation via hairpins/riboswitches.
    • 3' UTR: Stability elements, localization "zip codes," and poly(A) tail.
  • The Splicing Landscape:
    • The 20,000 human genes produce ~100,000 transcripts through alternative splicing.
    • Average transcribed region is 27 kb27\,kb, while the average gene is 1.3 kb1.3\,kb (~5%5\% usage).
  • Non-coding RNAs (ncRNA):
    • lncRNA: Long non-coding RNAs; act as scaffolds or enhancers for chromatin regulation.
    • miRNA: MicroRNAs (~21 nt21\,nt); regulate post-transcriptional expression via down-regulation. Conserved across species; involved in organ development and disease (e.g., 25 linked to pancreatic cancer).
    • siRNA: Small interfering RNAs; respond to foreign RNA (viruses/transposons). Processed by Dicer and integrated into RISC.
  • RNA Interference (RNAi): Heritable in C.elegansC. elegans for over 100 generations. Environmental responses (starvation, trauma) can be passed via RNAi to offspring.

Chromosome Structure and the Cell Cycle

  • Centromeres: One per chromosome; attachment point for kinetochores. Bound by the histone H3 variant CENP−ACENP-A.
  • Telomeres: Single-stranded ends with TTAGGG repeats. Protected by telomerase.
  • The Cell Cycle:
    • G1: Growth and preparation.
    • S (Synthesis): DNA replication and histone synthesis (35x increase in histone mRNA). Sister chromatids are joined by cohesin.
    • G2: Final preparation for division.
    • M (Mitosis): Chromosome segregation.
  • Cell Cycle Checkpoints: Regulated by cyclins and cyclin-dependent kinases (CDKsCDKs). Phosphorylation of retinoblastoma protein (RBRB) allows progression. p53 acts as a DNA damage sensor.
  • Mitosis Mechanism:
    • Prophase: Condensin added for condensation.
    • Metaphase: Alignment at the plate; spindle attachment.
    • Anaphase: Cohesin cleaved by separase, allowing separation.
    • Telophase: Decondensation and cytokinesis.

Meiosis and Recombination

  • Process: Two rounds of segregation (2n→4n→2n→n2n \rightarrow 4n \rightarrow 2n \rightarrow n).
  • Meiosis I: Homologous chromosomes pair. Crossing over (recombination) is essential for tension and segregation.
    • Sperm: ~26 crossovers.
    • Eggs: ~45 crossovers.
  • Female Specifics: Meiosis begins before birth, arrests at Prophase I until puberty, then arrests at Metaphase II until fertilization. Cohesin deterioration with age leads to aneuploidy.
  • Meiotic Drive: "Selfish genes" that cheat to increase transmission (e.g., Segregation Distortion in DrosophilaDrosophila with 99%99\% inheritance).
  • Shugoshin: A protein ("guardian spirit") that protects centromeric cohesin from cleavage during Meiosis I by recruiting a phosphatase.

The Nucleosome: Building Blocks of Chromatin

  • Structure: DNA (147 bp147\,bp) wraps 1.65 times1.65\,times around an octamer of eight histones: two each of H2AH2A, H2BH2B, H3H3, and H4H4.
  • Compaction: Provides ~6-fold compaction.
  • Linker DNA: Connects nucleosomes; ~20-60 bp long (variable by species: Humans 38-53 bp, Sea Urchin 110 bp).
  • Histone Proteins: Small, rich in lysine and arginine (++ charge). Histone fold domain mediates assembly.
  • Interactions: Non-sequence specific. Histones make 14 contacts with the minor groove and ~40 hydrogen bonds with the phosphodiester backbone.
  • Superhelicity: Nucleosomes induce negative superhelicity (ΔLk=−1.2\Delta Lk = -1.2 per nucleosome), storing energy that favors DNA unwinding.
  • Histone Variants:
    • H2A.XH2A.X: Phosphorylated during double-strand breaks.
    • MacroH2AMacroH2A: Silences the X chromosome.
    • CENP−ACENP-A: Replaces H3 in centromeres.

Higher-Order Chromatin and Regulation

  • The 30-nm Fiber: Nucleosomes pack further; requires Histone H1H1 (linker histone).
  • Models: Solenoid (superhelix, 6 nucleosomes/turn) and Zigzag (longer linker DNA passes through the center). Data mostly supports the Solenoid model.
  • Euchromatin vs. Heterochromatin:
    • Euchromatin: 10-nm fiber ("beads on a string"), open, high transcription.
    • Heterochromatin: 30-nm fiber, condensed, silent.
  • Nucleosome Remodeling: Complexes use ATP to slide, remove, or exchange nucleosomes.
  • Histone Tail Modifications: The "Histone Code."
    • Acetylation: Performed by HATs (Histone Acetyltransferases); loosens chromatin/activates genes. Recognized by Bromodomains.
    • Methylation: Performed by HMTs (Histone Methyltransferases); usually silences/represses. Recognized by Chromodomains.
  • Inheritance of Marks: During replication, nucleosomes are reassembled using chaperones like CAF-1 (interacts with PCNA) and NAP-1. Humans likely show semiconservative inheritance of H3/H4H3/H4 marks.

Epigenetics and Environmental Influence

  • Definition: Changes in gene expression resulting from environmental triggers, not DNA sequence changes.
  • Global Reset: Most marks are removed in the zygote (protamines replace histones in sperm), but some persist for generations.
  • Clinical and Environmental Examples:
    • Dutch Hunger Winter: Offspring of starving mothers showed increased susceptibility to obesity and metabolic disorders.
    • Stress: Maternal stress (e.g., 1998 ice storm) linked to altered DNA methylation in immune genes of children.
    • Trauma: Paternal stress and childhood abuse are associated with specific sperm DNA methylation patterns.
    • Microbial Influence: Gut bacteria can activate HDAC3HDAC3, regulating host fat absorption.
    • Viral Subversion: Influenza NS1 protein mimics histone H3 to block antiviral gene expression.
  • Modern Genetic Formula: Genotype + Environment + Triggers + Chance →\rightarrow Phenotype.