Histone Modifications and DNA Methylation

### Histone Modifications

• Acetylation of lysine residues on histones eliminates the positive charge, making the DNA less tightly bound.
• Acetylation facilitates transcription.

DNA Methylation

• Covalent attachment of methyl groups (-CH3) to DNA.
•  Carried out by DNA methyltransferase.
• Common in some eukaryotic species, but not all (e.g., yeast and Drosophila have little DNA methylation, while vertebrates and plants have abundant DNA methylation).
• In mammals, approximately 2 to 7% of DNA is methylated.
• DNA methylation typically inhibits eukaryotic gene transcription.

CpG Islands

• Found in vertebrates and plants, near gene promoters.
• 1,000 to 2,000 nucleotides long and contain a high number of CpG sites.
• In housekeeping genes, CpG islands are unmethylated, leading to gene expression in most cell types.
• In tissue-specific genes, methylation of CpG islands can silence gene expression.
• Methylation may influence the binding of transcription factors.
•  Methyl-CpG-binding proteins can recruit factors that compact chromatin.

Inheritance of DNA Methylation

• Methylated DNA sequences are inherited during cell division.
•   May explain genomic imprinting, where specific genes are methylated in gametes from either the female or male parent.
• The pattern of one copy of the gene being methylated and the other not is maintained in offspring.

### Gene Activation

• A series of events leading to gene transcription to produce an RNA molecule.
• Requires regulatory transcription factors (activators) binding to enhancers.
• Activators recruit coactivators – chromatin remodeling complexes and histone-modifying enzymes – to the promoter region.
• An RNA polymerase binds to the core promoter to form a preinitiation complex.
• RNA polymerase then elongates the transcript.

### Nucleosome Arrangement

• A nucleosome-free region (NFR) exists at the beginning and end of many eukaryotic genes.
• Precise positioning of nucleosomes near the beginning and end, but a less regular distribution elsewhere.

Transcriptional Activation

• Involves changes in nucleosome position and composition, as well as histone modifications.
•   Activators recruit chromatin remodeling complexes and histone-modifying enzymes.

Histone Variants

•  Five types of histone genes, moderately repetitive (H1, H2A, H2B, H3, and H4).
•  Human genome contains over 70 histone genes.
• Most code for standard histones.
•  Some accumulate mutations, altering the amino acid sequence, creating histone variants.

Histone Variants and Chromatin Structure

• Incorporated into a subset of nucleosomes to create specialized chromatin.
• Some variants promote an open chromatin conformation, while others promote a closed conformation.
•  Changes in the composition of histone variants can regulate gene transcription.
• Other functions include DNA repair and chromosome segregation.

Histone Code

• Over 50 enzymes covalently modify the amino terminal tails of histones.
•  These modifications affect transcription level by:
  • Influencing interactions between DNA and histone proteins.
  • Creating patterns that are recognizable by proteins (the histone code).
• The histone code provides binding sites for proteins, influencing chromatin structure and ultimately transcription.

 Histone Modifications and Structure

• Acetylation, methylation and phosphorylation are common histone modifications.
• Certain protein codes recognize specific combinations of modifications, such as phosphorylation of serine at position 1 in H2A and acetylation of lysine at the 5th and 8th positions in H4.

Gene Regulation in Eukaryotes

• Gene expression is the process of using genetic information to synthesize RNA and proteins, ultimately affecting cell properties and organism phenotypes.
• Gene regulation refers to the variable levels of gene expression under different conditions.
• The regulation of gene expression is crucial for various biological processes:
  • Responding to environmental changes like nutrient availability and stress.
  • Generating diverse cell types in multicellular organisms.
  • Facilitating developmental changes, as some genes are exclusively expressed during embryonic stages or adulthood.

Levels of Gene Regulation

• Transcription:
  • Regulatory transcription factors activate or inhibit transcription.
  • Nucleosome arrangement and composition influence gene expression.
  • DNA methylation can repress transcription.
• RNA modification:
  • Alternative splicing and RNA editing modify RNA transcripts.
• Translation:
  • Proteins control translation and mRNA degradation.
  • RNA interference regulates gene expression post-transcriptionally.
• Post-translation:
  • Feedback inhibition and covalent modifications regulate protein activity.

Combinatorial Control of Gene Expression

• Most eukaryotic genes are regulated by multiple factors, working in a combinatorial manner.
• Factors contributing to combinatorial control:
  • Activators stimulate transcription.
  • Repressors inhibit transcription.
  • Activators and repressors can be modulated by small effector molecules, protein interactions, and covalent modifications.
  • Regulatory proteins may alter nucleosome structures near promoters.
  • DNA methylation can repress transcription.

Histone Variants and the Histone Code

• Histone variants are incorporated into nucleosomes, creating specialized chromatin structures.
• Some variants promote open chromatin, facilitating transcription, while others favor closed chromatin, restricting transcription.
• The histone code refers to the pattern of covalent modifications on histone tails, influencing DNA-histone interactions and serving as binding sites for proteins that regulate chromatin structure and transcription.

Histone Modifications

• Common histone modifications include acetylation, methylation, and phosphorylation.
• These modifications alter nucleosome structure and affect gene expression.
• For example, phosphorylation of serine at position 1 in H2A and acetylation of lysine at positions 5 and 8 in H4 are recognized by specific proteins, influencing chromatin structure and transcription.

Steroid Hormone Regulation

• Steroid hormones bind to regulatory transcription factors called steroid receptors, altering their activity.
• Steroid hormones are produced by endocrine glands, secreted into the bloodstream, and taken up by target cells.
• Examples of steroid hormones:
  • Glucocorticoids: Influence nutrient metabolism in most cells.
  • Gonadocorticoids: Influence the growth and function of gonads (e.g., estrogen, testosterone).

Glucocorticoid Hormone Action

• Glucocorticoid hormones bind to glucocorticoid receptors, which dimerize and enter the nucleus.
• They bind to glucocorticoid response elements (GREs) located near genes, activating transcription.
• GREs consist of two consensus sequences: 5’-AGRACA-3’ and 3’-TCYTGT-5’.

CREB Protein and cAMP Signaling

• CREB protein binds to cAMP response elements (CREs) in response to cAMP signaling.
• CREs have the consensus sequence: 5’-TGACGTCA-3’ and 3’-ACTGCAGT-5’.
• Activation of CREB protein occurs through phosphorylation by protein kinase A, stimulated by increased cAMP levels.
• Phosphorylated CREB binds to the coactivator CREB-binding protein (CBP), enhancing transcription of the adjacent gene.

Gene Repression

• Gene repression inhibits transcription, reducing RNA synthesis.
• Repressors bind directly to DNA sequences, often within enhancers, preventing transcription.
• Repressors interact with corepressors to inhibit gene activation.
• Repression can be short-term, directly blocking activation steps, or long-term, as in heterochromatin formation, which silences genes permanently.

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

• ChIP-Seq maps locations of specific nucleosomes, histone variants, and histone modifications within a genome.
• It involves immunoprecipitation of chromatin using antibodies against specific proteins, followed by DNA sequencing.

Comparison of Transcriptional Regulation in Bacteria, Archaea, and Eukaryotes

• Gene organization:
  • Bacteria: Often in operons.
  • Archaea: Often in operons.
  • Eukaryotes: Usually in single genes, with exceptions like operons in C. elegans.
• Location of regulatory elements:
  • Bacteria: Operators are typically located near promoters.
  • Archaea: Operators are typically located near promoters.
  • Eukaryotes: Enhancers are located at more distant sites from promoters, requiring DNA looping to bring them close together.
• Transcription factors:
  • Bacteria: Sigma factor is needed for promoter recognition.
  • Archaea: Have homologs of eukaryotic TBP (TATA-binding protein) and subunits of TFIIB and TFIIE.
  • Eukaryotes: Six general transcription factors and mediator assemble at the core promoter.
• Activators and repressors:
  • Bacteria: Bind to operator sites, interacting directly with RNA polymerase or altering DNA structure.
  • Archaea: Bind to operator sites, similar to bacteria.
  • Eukaryotes: Bind to enhancers, recruiting coactivator complexes that modify chromatin structure and interact with GTFs and mediator.
• DNA methylation:
  • Bacteria: Yes.
  • Archaea: Yes.
  • Eukaryotes: Yes.
• Riboswitches:
  • Bacteria: Yes.
  • Archaea: Yes.
  • Eukaryotes: Yes (but not in animals).