BIOB11 L2

Structure of Genomes

  • Eukaryotes (Animals, plants, and fungi):

    • Genomic double-stranded DNA is found in the nucleus.

    • Additional DNA in mitochondria, chloroplasts, pathogens, etc.

  • Prokaryotic cells (Archaea and bacteria):

    • Genomic double-stranded DNA is found in the nucleoid.

    • Lots of horizontal gene transfer between species.

  • Viruses: Double or single stranded DNA or RNA

Comparing Genomes: Bacteria vs. Human

  • Over 200 gene families are common to all living organisms.

  • Human chromosomes are linear, with 23 pairs (diploid).

Genome Size

  • Genome size varies tremendously.

  • Size does NOT necessarily correlate with the number of genes or chromosomes.

  • Corn (maize) has 32k protein-coding genes but fewer base pairs and chromosomes than humans.

Eukaryotic Genomes

  • Non-coding DNA can make up a large percentage of eukaryotic genomes.

  • Non-coding DNA is often repetitive.

Composition of the Human Genome

  • The human genome consists of:

    • Protein-coding regions

    • Introns

    • Unique sequences

    • Repeated sequences (LINES, SINES, retroviral-like elements, DNA-only transposon "fossils", simple sequence repeats, segmental duplications)

Chromosomes and Genes

  • Non-coding sequences can exist within genes.

  • Introns are removed by RNA splicing after transcription.

  • Remaining protein-coding sequences are called exons.

Packaging the Eukaryotic Genome

  • Genome must be packaged to fit in the nucleus.

    • In humans, a diploid cell has approximately 6.4 billion base pairs of DNA in 46 chromosomes.

    • Length of 1 base pair = 0.34 nm, so 6.4 billion base pairs is approximately 2 meters.

    • Each base pair binds 6 water molecules, expanding volume.

    • All this has to fit in a nucleus that is 10 \mu m in diameter.

  • Packaging must be compact but also accessible to enzymes and regulatory factors.

DNA Supercoiling

  • Supercoiling is helpful to relieve strain.

  • Increases stability.

  • Makes DNA more compact.

  • Allows unwinding of sections.

DNA Structure: Supercoiling

  • Topoisomerases are protein enzymes that regulate/relieve supercoiling.

  • Positive supercoils: helix is overwound.

  • Negative supercoils: helix is underwound.

Packaging the Genome: Nucleosomes

  • Eukaryotic DNA associates with histones and other proteins to form chromatin.

  • Histones package DNA into repeating units called nucleosomes.

Chromatin Organization - Nucleosomes

  • Nucleosome: Repeating subunit of DNA + histones.

    • 8 histone proteins form a central core (octamer: two each of H2A, H2B, H3, and H4).

    • DNA is wound around the octamer core.

  • Overall, the packaging ratio of DNA in nucleosomes is 7:1 ((0.34 nm / bp \times 200 bp = 70 nm of DNA occupying only 10 nm).

Nucleosomes and Histones

  • Histones are highly conserved proteins rich in basic amino acids (Lys, Arg), resulting in a high positive charge that interacts easily with the negative charge of DNA.

Chromatin Organization - Nucleosomes

  • Histone dimer formation is mediated by the C-terminal domain histone fold (histone handshake: H2A with H2B, H3 with H4).

  • Each histone has an N-terminal histone tail that protrudes outward and can be subject to covalent modifications.

Chromatin Organization - Histone H1

  • Histone H1 (linker histone) binds linker DNA that connects one nucleosome to another.

  • Regulates how tightly these nucleosomes pack together.

  • Without histone H1, nucleosomes look like ‘beads on a string’.

Higher Levels of Chromatin Organization – 30 nm Fiber

  • Histone H1 can line up nucleosomes end-to-end into two stacks.

  • This “30 nm fiber” increases the DNA-packaging ratio 6-fold further (overall now approximately 40-fold).

Higher Levels of Chromatin Structure – Looped Domains

  • 30 nm fiber may further be gathered into a series of large loops.

  • These loops are attached to protein scaffolds.

Levels of Chromatin Organization

  • As cells prepare for mitosis, looped domains get further compacted.

  • DNA-packaging ratio in a mitotic chromosome is 10,000-fold.

  • Overall, the nucleus with a 10 \mu m diameter can pack 200,000 times this length of DNA!

Heterochromatin and Euchromatin

  • Euchromatin: DNA that is less compacted and functionally active (accessible for protein binding and transcription). Genes here can be expressed.

  • Heterochromatin: DNA that is highly compacted and has little to no functional activity.

Types of Heterochromatin

  • Constitutive heterochromatin

    • Permanently silenced DNA.

    • Includes regions around telomeres and centromeres.

    • Contains DNA repeats and few genes.

  • Facultative heterochromatin

    • Inactivated during certain phases of an organism’s life.

Facultative Heterochromatin Example

  • One of the X chromosomes in mammalian females is mostly silenced to ensure that males and females have the same number of active X chromosomes.

  • The inactive X chromosome is condensed as heterochromatin (Barr body).

Facultative Heterochromatin – X Chromosome Inactivation

  • X inactivation occurs early during embryonic development.

  • Random process where either maternal or paternal X chromosome is inactivated in any given cell.

  • Maintained: As the cell divides, the same X stays inactivated in daughter cells!

Facultative Heterochromatin – X Chromosome Inactivation (Visible Example)

  • Coat patterns on the backs of heterozygous female Calico cats.

Epigenetic Inheritance (Epigenetics)

  • Epigenetics: Covalent modifications (e.g., methylation) of DNA and histones.

  • Influences heterochromatin vs. euchromatin formation.

  • Epigenetic regulation of gene expression can be inherited by daughter cells during cell division without changes to the sequence of nucleotides in DNA.

The Histone Code

  • Covalent modifications of histone tails can disrupt or stabilize nucleosome assemblages.

  • Generally, acetylation leads to a more open structure and more transcription, whereas the majority of methylation causes compaction/repression.

Regulating Chromatin Structure - Histone Tail Modifications

  • Histone acetyltransferases (HATs) acetylate histone proteins by transferring an acetyl group from acetyl-CoA to specific lysine residues.

  • Histone deacetylases (HDACs) remove the acetyl group.

  • Histone methyltransferases (HMTs) add methyl group(s) (1, 2, or 3) to lysine or arginine residues.

  • Histone demethylases remove methyl groups.

Epigenetic regulation: DNA methylation

  • Methyl group can be attached directly to cytosine in DNA.

  • DNA methylation works together with the histone code.

  • Proteins that bind to methylated DNA can recruit enzymes involved in modifying histone tails.

  • Methylation of cytosine can prevent proteins from binding to DNA sequences (often preventing the initiation of transcription).

Epigenetic “memory” of DNA methylation patterns

  • DNA methyltransferase can add the methyl group to DNA at sites where a C is followed by a G (reading 5’ to 3’ = “CpG”) creating an epigenetic marker on the DNA.

  • DNA demethylases can remove these methyl groups.

  • DNA is methylated as it is replicated so that methylation patterns can be passed on to daughter cells (The symmetry of CpG on both strands is critical for this!).

Maintaining Epigenetic Regulation

  • Silencing of genes by heterochromatin formation occurs in regions (“position effect”) and is maintained in replicated DNA.

Epigenetic Regulation from Gametes to Offspring

  • Methylation patterns are copied to newly synthesized DNA and so can be inherited during cell division, but are mostly rewritten during the initiation of offspring development.

  • Some (very few) methylation patterns are passed on from parents to offspring (called genomic imprinting).

Reading the Histone Code

  • Histone modifications are recognized by specific proteins that are often part of multi-protein complexes.

  • Reader complex: “Reads” the histone code and positions and activates enzymes (“writers”) that can act on the adjacent DNA/histones.

Maintaining Heterochromatin After DNA Replication

  • The epigenetic signals that regulate chromatin are propagated through space (along chromosome) and time (during cell division)!

Enzymes Contributing to a More Condensed (Heterochromatin) State

  • HDACs (histone deacetylases).

  • Histone methyltransferases.

  • DNA methyltransferases.
    *Initial recruitment of these enzymes often occurs through a transcription factor protein binding to a specific DNA sequence.

Mechanisms of Barrier Action

  • Barrier proteins can bind to barrier sequences and create physical obstacles or actively recruit opposing chromatin-modifying enzymes.

  • Chromatin becomes separated into domains with different transcriptional activation/regulation.