Chromosome packaging
-End to end length of DNA in diploid human cell ~2m
-nucleus diameter ~5µm
~10 000x reduction in length
Chromosome features when stained with Giemsa
Heterochromatin -dark bands, condensed DNA -Not transcriptionally active Euchromatin -Light regions, less compact DNA -transcriptionally active regions Increased darkness=increased condensation=decreased transcriptional activity
Telomeres
Sequence (TTAGGG)n
n=800-2500
-hTERT: human telomerase reverse transcriptase, is increased in adult + embryonic stem cells and cancer
Functional requirements of DNA compaction
- highly organized
- allow access for factors that regulate replication
- Allow access for factors that regulate transcription
What is chromatin
-DNA + protein making up chromosomes
Nucleosomes
- unit of chromatin
- DNA wrapped around histone core
- 1.67 turns of DNA around histone, 147 BP
- H1 Subunit: linker histone
- with H1 Linker: 168 BP
Histone properties
- assemble into octameric complexes
- pos charge side chain neutralize neg charged DNA backbone (acts like a salt)
- high % of Lys and Arg residues in histones (20-30%), highly conserved over eukaryotes
Histone Octamer
- “core” histone subunits
- H2A, H2B form a dimer. Are found 2x in each octamer
- H3, H4 form a tetramer
- requires the presence of DNA and chaperone proteins to assemble
Histone Fold motif
- Straight 10 bp segment joined by bends
- motif is 3 a-helices, linked by 2 short loops
DNA sequence and nucleosome binding
- 1 turn of double helix = 10BP
- DNA compaction A-T>G-C (b/c weaker H-bonds)
- regions that interact best with histone octamers have A-T pairs 10 BP apart, offset by G-C pairs positioned in between
- A-T pairings in the region where the minor groove of the pairing is facing the histone allows for it to be compressed, bc DNA is naturally bent at A-T pairs
N-Terminal histone tails
- Flexible tails
- exit core via alignment of minor grooves every 20 BP
- mediate higher order chromatin structure
- pos charge tails allow for better compaction
- Tail modifications impact nucleosome interactions, changing the degree of chromatin compaction
- Increased interactions=increased DNA access=Increased transcription
H1 “linker” histone
-one linker per octamer
-protects additional 21 BP
binding sites:
-DNA linker region (free end)
-central region of nucleosome DNA
-fixes DNA entry/exit angles (no linker= free DNA ends)
-provides extra 6-7x compaction of nucleosomes
-compaction by nucleosomes=6-7x, compaction by H1= 6-7x, total ~36-42x
30nm filament
- after binding of H1, nucleosomes condense into filament with 30nm width
- can form without H1
- can’t form without N-term tails of core histones
- seen in vitro, not known if seen in vivo
Requirements of DNA compaction system
- Dynamic
- rapid changes in condensation so replication/transcription can occur when needed - Modifiable
- globally (ie. replication)
- locally (ie. transcription of a specific gene) - Responsive
- respond to modification enzymes that alter state of DNA condensation
- specific regions need to be recognizable by the enzymes
What classes of enzymes allow regulation of DNA compaction to be achieved
- Chromatin remodelling complexes
- these change location and variant content of octamers - Histone modifying enzymes
- they modify the N-term regions (ie. acetylate lysine)
