MBB 267 Week 9: Mitchel 4 Flashcards
What is chromatin?
Chromatin is DNA that is packaged by histones, non-histone proteins and RNA
What is chromatin fundamentally made out of?
Nucleosomes
What happens to chromatin when extracted from;
- low salt conditions?
- High salt condition?
Basically:
- Chromatin extracted from nuclei under low salt conditions and in the absence of Mg2+ has a necklace-like appearance often described as “beads on a string”. The particles are known as nucleosomes, and are joined together by linker DNA. Nucleosomes consist of DNA wound around a core of histone proteins. Nucleosomes are ~ 10 nm in diameter.
- When extracted under more physiological conditions, chromatin is observed as more compact 30 nm fibres. By changing the conditions, chromatin can be dynamically reorganised between these two forms.
How are nucleosomes isolated?
Limited nuclease digestion of chromatin allows digestion of the linker DNA and allows the isolation of nucleosome particles
What is the structure of a nucleosome particle?
The core structure of nucleosome complexes have an octameric structure, consisting of two copies of the histone proteins H2A, H2B, H3 and H4 that are arranged as H2A/H2B and H3/H4 dimers. The core histones comprise a central folded domain and have flexible N-terminal regions known as “tails” (H2A and H2B also have short C-terminal “tails”). The paired subunits are interleaved with each other in a “handshake” like interaction. The DNA is wrapped around the core complex, 147 base-pairs making one and two third (~1.666) turns in a left-handed direction. The core histones make multiple contacts with the phosphate backbone of the surrounding DNA but the contacts can be dynamic, allowing nucleosomes to be moved along the DNA. The histone tails extend outside of the structure and mediate interactions between histones and with other proteins. The length of linker DNA joining two adjacent nucleosomes varies between 10 and 100 base-pairs but is a multiple of 10 nucleotides (one helical turn of the DNA). The histone protein H1 interacts with the DNA as it enters and exits the nucleosome. The core histones are highly conserved but some nucleosomes contain histone variants, such as H2AX, which is incorporated into nucleosomes at sites of DNA repair, H3.3 which is the predominant form of H3 in nondividing cells, and CENP-A, which is a variant of H3 found in centromeres.
Explain the structure of the 30nm nucleosome fibres?
Packaging of the 10 nm fibre into the 30 nm fibre requires linker histone H1 and the tails of the core histones. Two models have been proposed for structural organisation of the 30 nm fibre, depending on whether the chain of nucleosomes is wound into a single coil (the solenoid model) or whether the linker DNA stretches across a two-stranded left-handed double helix of nucleosomes (the zig-zag ribbon model). The structures are based on reconstituted DNA/nucleosome complexes but both models may reflect natural structures
What is a chromosome territory?
each individual chromosome is localised to a restricted subregion of the cell nucleus
What happens to chromosome territories during transcription?
decondensation of chromosome territories therefore, transcription can only occur at decondensed areas of the chromosome.
actively transcribed genes can be found in DNA that is looped out.
What are transcriptional factories?
actively transcribed genes are associated with RNA polymerase and transcription factors at specific sites known as transcription factories.
What are TADs?
Topologically associated domains (TADs) are spatially restricted regions of the chromosome that are coordinately regulated in terms of gene expression and timing of replication.
How do TADs work?
TAD boundaries are established by CTCF and cohesin and are flanked by lamina associated domains (LADs) that contain transcriptionally silenced DNA. This allows enhancers/silencers to interact within TADs.
How are histones modified?
Core Histone Tails are Post-translationally Modified. Histones have flexible tails. The tails can be post-translationally modified through acetylation (lysine), methylation (lysine,arginine), phosphorylation (serine,threonine) and ubiquitylation (lysine). Some modifications are mutually exclusive (methylation of lysine residue blocks acetylation), whereas other modifications are mutually dependent (e.g. ubiquitylation of H2B is required for methylation of H3K4). Post-translational modifications of histones act as epigenetic markers to influence chromatin structure and gene expression.
How is lysine modified in histones?
Many important histone modifications involve the e-amino group of lysine side chains (acetylation, methylation, ubiquitylation). Histone acetyltransferases (HATs) add acetyl groups to the epsilon amino group of lysine residues. Acetylation neutralises the positive charge of the amino group. This modification is readily reversed by histone deacetylase complexes (HDACs). Methylation of lysine side chains by histone methyltransferases (HMTs) prevents acetylation and does not affect the positive charge. Methyl groups were thought to be stable but have been shown to be slowly removed by recently discovered lysine-specific demethylases (LSDs). These modifications affect interactions between nucleosomes and with other factors
