Lecture 2: Regulation by Chromatin 🧬📜

Focus: This lecture explores how chromatin structure, particularly nucleosomes and their modifications, along with chromatin remodeling complexes, regulate gene expression in eukaryotes.

Insulators: Defining Genomic Boundaries

Insulators are DNA elements that play crucial roles in organizing the genome and regulating gene expression. They have two main functions:

  • Enhancer-Blocking Activity: Insulators can prevent enhancers (or silencers) from inappropriately acting on the promoters of neighboring genes that are not their intended targets.

  • Chromatin Boundary Markers: They mark the borders between different chromatin domains, such as separating regions of condensed heterochromatin from open euchromatin.

    • Example: In the human β-globin gene locus, insulators flank the gene cluster, separating it from neighboring genes like the folate receptor gene (often in heterochromatin) and the odorant receptor gene cluster. The β-globin locus itself contains a Locus Control Region (LCR) with DNase I hypersensitive (HS) sites (HS1-HS5), which is crucial for the correct developmental expression of the embryonic (ε), fetal (Gγ, Aγ), and adult (δ, β) globin genes.

Chromatin Modification Complexes and Histone Tails 🔧

The N-terminal tails of the core histones (H2A, H2B, H3, and H4) are key targets for regulation and are subject to a wide range of post-translational modifications. These modifications include:

  • Methylation

  • Acetylation

  • Phosphorylation

  • Ubiquitylation

Chromatin modification complexes are multiprotein assemblies that enzymatically add or remove these modifications.

  • They function as master on/off switches that determine whether a gene is active or inactive.

  • These modifications contribute to epigenetic regulation, as they are heritable changes not encoded in the DNA sequence itself.

  • The patterns of histone modifications serve as recognition landmarks for other proteins that bind to chromatin and influence its structure and function.

  • The levels of specific histone "marks" are dynamically maintained by the balanced activities of modifying enzymes (that add marks) and de-modifying enzymes (that remove marks).

Key Histone Modifying Enzymes and Their Effects:

  1. Histone Acetyltransferases (HATs) and Deacetylases (HDACs):

    • HATs add acetyl groups to lysine residues on histone tails.

    • HDACs remove these acetyl groups.

    • Histone acetylation generally neutralizes the positive charge of lysine, weakening the interaction between histones and DNA, and is usually associated with active chromatin (euchromatin) and gene activation.

    • Acetylated lysine residues create binding sites for proteins containing bromodomains.

  2. Histone Methyltransferases (HMTs) and Demethylases:

    • HMTs add methyl groups to lysine and arginine residues on histone tails.

    • Histone demethylases (e.g., LSD1) remove methyl groups.

    • Histone methylation can be associated with both transcriptional activation and repression, depending on the specific residue methylated and the number of methyl groups added (mono-, di-, or tri-methylation). For example, H3K4me3 is often associated with active promoters, while H3K9me3 and H3K27me3 are linked to heterochromatin and repression.

    • Chromodomains are protein motifs that specifically bind to methylated lysine residues and are often found in proteins involved in transcriptional silencing. DNA methyltransferases (DNMTs) can also contribute to forming heterochromatin in conjunction with repressive histone methyl marks.

  3. Ubiquitin-Conjugating Enzymes and Isopeptidases:

    • Ubiquitin-conjugating enzymes add a single ubiquitin molecule (monoubiquitylation) to a lysine residue.

    • Isopeptidases remove ubiquitin.

    • Monoubiquitylation of histone H2B can be associated with either gene activation or silencing, depending on the context.

    • Monoubiquitylation of the linker histone H1 can lead to its release from DNA, promoting gene activation.

  4. Kinases and Phosphatases:

    • Kinases add a phosphate group to serine or threonine amino acids on histone tails, introducing a negative charge.

    • Phosphatases remove these phosphate groups.

    • Phosphorylation of histone H3 (e.g., H3S10ph) or the linker histone H1 is often associated with the activation of specific genes and chromosome condensation during mitosis.

Nucleosomes and Transcription: How RNA Polymerase Accesses DNA

The packaging of DNA into nucleosomes presents a barrier to the transcription machinery. Several models describe how RNA polymerase navigates through chromatin:

  • Nucleosome Mobilisation Model:

    1. RNA Polymerase II initially transcribes the more accessible linker DNA between nucleosomes.

    2. As it approaches a nucleosome, there is a partial dissociation of DNA from the histone octamer, allowing the polymerase to proceed.

    3. The DNA behind the RNA Polymerase transiently re-binds to the histones.

    4. After about 60bp of transcription through the nucleosomal DNA, the histones may fully dissociate.

    5. Histones then reassociate to form a nucleosome behind the transcribing polymerase.

  • Histone Dimer Depletion Model (FACT Complex):

    • The FACT (FAcilitates Chromatin Transcription) complex plays a key role.

    • FACT displaces one H2A/H2B dimer from the nucleosome as RNA Polymerase II approaches, leaving a more open hexasome structure (H3/H4 tetramer + one H2A/H2B dimer).

    • FACT acts as a histone chaperone, holding onto the displaced H2A/H2B dimer and replacing it after the polymerase has passed, thus reassembling the nucleosome.

  • Nucleosome Positioning:

    • Nucleosomes are not randomly distributed but are often specifically positioned relative to gene features like promoters and TF binding sites.

    • For example, "growth genes" might have a different nucleosome landscape (e.g., around poly(dA:dT) tracts) compared to "stress genes" (which might have nucleosomes positioned over TATA boxes).

  • Inheritance of Histone Modifications:

    • Histone modifications can be stable and inherited during DNA replication. Parental H3-H4 tetramers, with their existing modifications, are distributed to daughter DNA strands. These modified histones can then recruit enzymes that catalyze the same modifications on newly synthesized and deposited histones, helping to propagate the parental chromatin state.

The "Histone Code" Hypothesis

  • The idea that specific combinations of histone modifications act as a "code" that is read by other proteins to bring about distinct downstream events.

  • Bromodomains are protein motifs that specifically recognize and bind to acetylated lysine residues on histone tails.

  • Chromodomains are protein motifs that specifically recognize and bind to methylated lysine residues. These interactions recruit other proteins and complexes that further modify chromatin or directly affect transcription.

Transcriptional Co-activators and Co-repressors

These are proteins or protein complexes that increase (co-activators) or decrease (co-repressors) transcriptional activity without binding to DNA directly.

  • They typically bind to DNA-bound transcription factors.

  • They alter chromatin structure, either:

    • Directly: Possessing enzymatic activity themselves (e.g., being a HAT or HDAC).

    • Indirectly: Recruiting other proteins or complexes with enzymatic activity.

  • Two main classes of such co-regulators that act on chromatin:

    1. Chromatin modification complexes (as discussed above, e.g., HATs, HMTs).

    2. Chromatin remodeling complexes.

Chromatin Remodeling Complexes 🌀

These large, multi-protein complexes use the energy from ATP hydrolysis to change the contacts between histones and DNA, thereby altering nucleosome structure and accessibility. This is an active process.

  • They allow transcription factors and the general transcription machinery to bind to DNA regulatory elements.

  • They can induce at least four different changes in chromatin structure:

    • Nucleosome sliding: Moving the histone octamer along the DNA.

    • Remodeled nucleosomes: Altering the conformation of the nucleosome to make DNA more accessible without fully displacing the octamer.

    • Nucleosome displacement/ejection: Removing the histone octamer from the DNA.

    • Nucleosome replacement: Exchanging canonical histones with histone variants (e.g., H2A.Z).

  • Experimental techniques like DNase I digestion followed by sequencing (DNase-seq) can map nucleosome positions by identifying DNA regions protected by nucleosomes. Such studies show that chromatin binding proteins position nucleosomes at relatively regular positions around key gene sequences (e.g., promoters, transcription factor binding sites). Remodeling can then regulate gene expression by enabling or restricting access to these sites.

Three Main Families of Chromatin Remodeling Complexes:

  1. SWI/SNF Complex Family:

    • The first chromatin remodeling complex to be characterized (from budding yeast), it's a large (~2 MDa) complex with at least 11 polypeptides. Many related factors exist from Drosophila to humans.

    • Primarily involved in nucleosome remodeling (altering nucleosome structure) and nucleosome displacement (ejecting nucleosomes from DNA).

    • Different SWI/SNF family members can regulate different genomic regions and interact with other remodeling complexes (e.g., affecting enhancers or promoters).

    • Plays a critical role in development and disease. For example, in stem cells, SWI/SNF complexes are involved in balancing self-renewal versus differentiation by activating differentiation genes. Mutations in SWI/SNF genes are found in a high number of cancers, where their inactivation can lead to impaired differentiation and tumorigenesis.

  2. ISWI (Imitation Switch) Complex Family:

    • Mainly responsible for nucleosome sliding, changing the position of histone octamers along the DNA without significantly perturbing their structure.

  3. SWR1 Complex Family:

    • Mediates histone replacement, specifically exchanging canonical H2A-H2B dimers within a nucleosome with dimers containing the histone variant H2A.Z (H2A.Z-H2B dimers).

    • This exchange can alter nucleosome stability and interactions, often converting chromatin to a more transcriptionally active state (euchromatin) from a silenced state (heterochromatin).

Summary

  • Nucleosomes, composed of a core of four histones (H2A, H2B, H3, H4) with DNA wrapped around them, and an external linker histone (H1), are fundamental to chromatin structure.

  • The binding of DNA to histones generally restricts access for RNA Polymerase II and other factors.

  • Post-translational modifications of histone tails (acetylation, methylation, phosphorylation, ubiquitylation) alter histone-DNA interactions and serve as binding sites for other regulatory proteins, influencing whether chromatin is open (euchromatin) or closed (heterochromatin) and thus affecting gene expression levels.

  • Chromatin is not static; it is actively remodeled by various ATP-dependent chromatin remodeling complexes (e.g., SWI/SNF, ISWI, SWR1 families) that can slide, eject, or restructure nucleosomes, or exchange histone variants, thereby regulating DNA accessibility and gene activity.

Required Reading

  • Alberts et al., 2008: Molecular Biology of the Cell, 5th Ed. Chap 4 (pp. 211-218).

  • Allison 2012: Fundamental Molecular Biology, 2nd Ed. Chap 5 (pp. 95-100 + 324-331).

  • Watson et al., 2008: Molecular Biology of the Gene, 6th Ed. Chap 7 (pp. 157-187).