Lecture 4 Part 3 Notes — Chromatin Remodeling, Heterochromatin, and the Histone Code

Nucleosome core particle and histone H1

  • Core histones and nucleosome composition:
    • A nucleosome consists of a histone octamer core: two copies each of the core histones 2\times H2A,\ 2\times H2B,\ 2\times H3,\ 2\times H4 per nucleosome.
  • Histone H1 (linker histone):
    • H1 is not a component of the histone core particle; it binds outside the core.
    • It binds nucleosomes at a 1:1 ratio: one per nucleosome, and is not necessarily present on every nucleosome.
    • Role: when bound, H1 constrains the movement of an additional 20\,\text{bp} of DNA and changes the exit angle of DNA from the nucleosome, enabling tighter chromatin packing.
    • In diagrams, H1-bound nucleosomes show a different exiting DNA angle than naked nucleosomes.
  • Nucleosome tails and packing:
    • Histone N-terminal tails project outward from the nucleosome.
    • Tails from adjacent nucleosomes reach out and contact one another, contributing to chromatin compaction alongside H1.

Nucleosome tails, covalent modifications, and the histone code

  • N-terminal tails are sites of covalent modification that influence nucleosome packing, protein binding, and even core nucleosome structure.
  • Covalent modifications (and enzymes):
    • Lysine acetylation (HATs) and deacetylation (HDACs).
    • Acetylation is catalyzed by histone acetyltransferases (HATs).
    • Deacetylation is catalyzed by histone deacetylases (HDACs).
    • Lysine methylation (HMTs) and demethylation (HDMs).
    • Methylation can be mono-, di-, or tri-methylated on lysines: me1, me2, me3.
    • Demethylation is carried out by histone demethylases (HDMs).
    • Serine phosphorylation and dephosphorylation (kinases and phosphatases).
    • Ubiquitination can also occur on lysines (not shown in every slide).
  • The modifications are dynamic and reversible, enabling chromatin to respond to signals.
  • The histone tails encode a combinatorial set of marks – the histone code – which is read by specific protein modules.

The histone code and reader/writer logic

  • Reader-writer model:
    • Writer enzymes place specific marks on histone tails.
    • Reader modules recognize specific histone marks (often in combination) and recruit other proteins.
    • Binding by reader complexes can recruit further writers, remodeling factors, or transcriptional regulators.
  • Reader complexes often recognize multiple marks rather than a single modification; they stably bind only when a chromatin region contains a compatible set of marks.
  • Consequences of reading marks:
    • Recruitment of chromatin remodelers, transcriptional activators or silencers, or writer complexes that propagate marks to neighboring nucleosomes.
  • Practical examples of marks on histone H3:
    • \mathrm{H3K_{9}me3} signals heterochromatin formation and gene silencing.
    • \mathrm{H3K{4}me3} together with \mathrm{H3K{9}ac} signals euchromatin formation and gene expression; acetylation at K9 can block methylation at the same site, and vice versa.
    • \mathrm{H3K_{27}me3} also signals gene silencing.
  • Key caveat: these interpretations are context-dependent and not read in isolation.

Remodeling and histone variant exchange

  • ATP-dependent chromatin remodeling complexes:
    • Reposition nucleosomes on DNA (nucleosome sliding) by binding to nucleosomes and DNA and using ATP hydrolysis to transiently loosen DNA-histone contacts.
    • Result: nucleosomes can be moved closer together or further apart along DNA, altering accessibility.
  • Yeast RSC chromatin remodeling complex (an example):
    • Shown as a dimer of ISW1 proteins in the diagram.
    • RSC can couple adjacent nucleosomes (yellow and orange in the figure) and influence transcription by:
    • Repressing transcription in some contexts,
    • Moving promoter-proximal nucleosomes,
    • Regulating RNA polymerase traversal through coding regions.
  • Beyond sliding, remodelers can exchange histone components:
    • They can remove histone octamers from DNA to replace with new octamers, potentially containing histone variants (e.g., \text{H2AZ} or \text{H2AX}).
    • They can also replace histone dimers within the core particle.
  • Histone chaperones (blue in the diagram) assist these exchanges by binding free histones or octamers that are not part of complete nucleosomes.
  • Histone variants:
    • H2AZ (also called H2A.Z) and H2AX are examples of histone variants involved in remodeling and DNA damage responses.
  • Gamma-H2AX:
    • At sites of DNA double-strand breaks, H2AX is phosphorylated to form gamma-H2AX (\gamma\mathrm{H2AX}).
    • Gamma-H2AX serves as a marker for DNA breaks and helps coordinate repair.

Euchromatin vs. heterochromatin and chromatin spreading

  • Definitions and locations:
    • Euchromatin: relaxed, open chromatin, typically gene-rich and transcriptionally active.
    • Heterochromatin: compact, transcriptionally silent chromatin, concentrated at telomeres and centromeres; associated with the nuclear lamina at the inner edge of the nucleus; contains relatively few genes.
  • Genomic distribution:
    • Heterochromatin accounts for about 10\% of the genome.
  • Dynamics:
    • Euchromatin and heterochromatin are dynamic and can be interconverted.
    • When euchromatin becomes heterochromatin, transcription in that region is silenced.
    • Heterochromatin can spread into adjacent euchromatin; barrier DNA sequences can block this spread.
  • Barrier DNA sequences:
    • These sequences prevent the spread of heterochromatin into neighboring euchromatin through different mechanisms (to be detailed below).
  • Diagrammatic example (left):
    • A region of heterochromatin (green) is adjacent to euchromatin containing five genes.
    • A barrier sequence (white) sits between the heterochromatin and euchromatin, blocking spread.
  • Consequences of barrier loss (translocation):
    • If barrier function is lost (e.g., via a chromosomal translocation placing heterochromatin next to a gene), the degree of heterochromatin spread into euchromatin can vary between cells.
    • As a result, different cells exhibit different silencing patterns for the same gene, leading to clonal variation in expression across the embryo.
  • Position effect variegation (PEV) (Drosophila example):
    • In fruit flies, a gene such as white (orange in the figure) is normally in euchromatin and expressed (red eyes).
    • Downstream heterochromatin is present but blocked by a barrier to spread in the wild type.
    • Chromosomal inversion can relocate barrier-free heterochromatin near the gene early in development; in some cells, heterochromatin spreads and silences the gene, while in others it does not, producing a mottled or variegated eye pattern in the adult.
    • This phenomenon is a classic demonstration of chromatin spreading and barrier function.
  • Takeaway:
    • Chromatin packing is dynamic; heterochromatin can spread through positive feedback loops of reader and writer activities, and barrier sequences can stop this spread.

How heterochromatin spreads: the reader-writer mechanism

  • Spreading model:
    • Start with an open chromatin region containing a regulatory DNA sequence bound by a regulatory protein (shown in purple).
    • This regulator recruits a histone-modifying writer enzyme to install a heterochromatin-associated mark (e.g., \mathrm{K9me3}).
    • A reader protein recognizes the new mark and recruits the same or a similar writer to the neighboring nucleosome, propagating the modification to adjacent nucleosomes.
    • The process iterates, creating a spreading wave of chromatin condensation.
  • Barrier sequences counteract spreading, depending on the type of barrier recruited proteins:
    • (A) Barrier protein tethering DNA to a nuclear structure (e.g., a nuclear pore): creates a physical barrier to heterochromatin spread.
    • (B) Barrier protein that covers several adjacent nucleosomes, making them inaccessible to enzymes that promote spread.
    • (C) Barrier enzyme recruited to the barrier sequence that erases histone modifications signaling heterochromatin formation.

Putting it together: the histone code, readers, writers, and barriers

  • The process of heterochromatin spread requires coordinated action of reader and writer complexes;
    • An initiating DNA-bound regulator recruits a writer to create a histone tail modification associated with heterochromatin (e.g., \mathrm{K9me3}).
    • A reader recognizes this mark and recruits a writer to neighboring nucleosomes, propagating the mark along the region.
    • The spreading produces a wave of chromatin condensation.
  • Barrier sequences block this spread via the three mechanisms described above, depending on the barrier proteins involved.

Practical examples and real-world relevance

  • Position effect variegation (PEV) in Drosophila demonstrates the consequences of chromatin spreading and barriers in a living organism:
    • Variegated eye color patterns arise from differential spreading of heterochromatin influencing gene expression in disparate cells.
  • The histone code has broad implications for:
    • Development: dynamic regulation of gene expression via chromatin states.
    • Genome stability: histone modifications participate in DNA repair signaling (e.g., gamma-H2AX).
    • Epigenetic inheritance: patterns of heterochromatin can be inherited by daughter cells.
  • Connection to nuclear architecture:
    • Heterochromatin localizes to the nuclear periphery and telomeres/centromeres, influencing genome organization and accessibility.
  • Final note:
    • These concepts set the stage for understanding the organization of chromosomes and chromatin in the nucleus, to be covered in the final part of the lecture.

Summary of key terms and concepts

  • Core histones: 2\times H2A,\ 2\times H2B,\ 2\times H3,\ 2\times H4
  • Linker histone: 1\times H1 per nucleosome (not part of the core octamer); constrains 20\,\text{bp} of DNA and alters DNA exit angle.
  • Histone tails and packing: tails contact neighboring nucleosomes; contribute to compaction with H1.
  • ATP-dependent chromatin remodelers: reposition nucleosomes (sliding), can remove octamers or exchange with variants; examples include yeast RSC complex (dimer of ISW1 proteins).
  • Histone variants: H2AZ, H2AX; gamma-H2AX marks DSBs for repair.
  • Euchromatin vs heterochromatin: open vs compact chromatin; telomeres/centromeres enriched in heterochromatin; ~10\% of genome.
  • Barrier DNA sequences: block heterochromatin spread via tethering to nuclear structures, masking nucleosomes, or erasing marks.
  • Histone code: combinatorial set of tail modifications (acetylation, methylation, phosphorylation, ubiquitination) read by protein readers and written by writers; acetylation vs methylation on lysines are mutually exclusive at the same site, e.g., \mathrm{K9\;me3} vs \mathrm{K9\;ac}.
  • Key examples of marks:
    • \mathrm{H3K_{9}me3}: heterochromatin, gene silencing.
    • \mathrm{H3K{4}me3} with \mathrm{H3K{9}ac}: euchromatin, gene expression.
    • \mathrm{H3K_{27}me3}: gene silencing.
  • Mechanistic takeaway: heterochromatin spreads via a reader-writer feedback loop; barrier sequences modulate the spread and genome organization.