Diapositivas

3. Chromatin and Nuclear Structure

Chloe Cavarretta Molecular Biology, Biotechnology and Bioinformatics Inglés 29 diapositivas

Diapositivas del conjunto (29)

Learning Outcomes: L8
- Understand the structure of chromosomes and the diversity of interphase chromatin
- Know about modifications that affect access and function
- Have an overview of the mechanisms for maintenance and change
- Appreciate the complex organization of a eukaryotic cell nucleus
Chromosome
- Chromatin is condensed to form a transmissable unit in mitosis = chromosome
- Every gene has a postcode on a chromosome eg CIZ1 is at 9q34.2
- Chromosome 9, q arm, position 34.2
Spectral Karotype Fluoresence
- Chromosome probed and identified using specific DNA in situ hybridisation probes that are chromosome specific
- Identify each chromosome in a different colour
- Or use probe specific for genes and used to locate a gene on a chromosome
Chromatin Cycle
- Every cell cycle chromosomes are condensed and decondensed
DNA Packaging
DNA Packing: Beads on a String
- DNA is the string and the beads are nucleosomes
- Nucelosome= octameric complex, 2 molecules each of H2A, H2B, H3, H4
- DNA is wound twice around each nucleosome core particle
- Intervening DNA= linker
DNA and Histones Made for Eachother
- 1. Toplogy
- 10.4bp of DNA per helical turn (twist)
- 146 nucleotides of dsDNA wrapped around each nucleosome in a relaxed state
- In vivo (bacterial cells with circular chromosomes or eukaryotic with linear), chromosomes are attached at intermediate locations to the nuclear matrix
- So that DNA molecules are tethered and are not free to add supercoils so remain in a relaxed, fixed state
DNA and Histones Made for Eachother
- 1, Topology
- Supercoiled DNA is the stressed state of DNA and occurs if a molecule has sufficient coils to create another twist (1 twist= writh of 1, 2 twists = writh of 2)
- More twists = positively supercoiled
- Less twists= negatively supercoiled
- DNA in the nucleus is thought to be negatively coiled however wrapped around a nucleosome it is a relaxed molecule- the process of unwinding the DNA form the nucleosome creates negative supercoils which is seen in DNA that is extracted from the nucleus (underwound DNA)
DNA and Histones Made for Eachother
- 2. Spacing
- A 5th linker histone (H1) seals the core particle - seals the 2 loops of DNA wrapped around to stabilise
- Linker DNA between nucleosomes is ~10-90bp long
- Nucleosome spacing is ~200bp
- Can visualise spacing between nucleosomes by using nucleases
- Extract chromatin from nucleus and digest with nuclease
- Fragments are separated according to size and DNA is fluorescently labelled so only see DNA and not nucleosomes
- See a 'nucleosome ladder'- pattern of DNA every 200bp
- 3. Charge
- Negative charge of DNA neutralised by positive charge of histones
- Chromatin complex= neutral, relaxed state complex
Topology Problems in Replication + Txn
- Processes that require access to the genome disrupt the relaxed state of chromatin:
- Create positive supercoils ahead of the replication fork/transcription complexes until they reach an anchor point (where attached to nuclear matrix)
- Topoisomerases relieve the stress to create a relaxed state
- They are highly enriched at anchor points
- Type I- cuts one strand, passes the other through then ligates it
- Type II- cuts both strands then passes an unbroken strand through - problem is that causes ds break
Topology Problems in Replication + Txn
DNA Packing: Solenoid
- Beads on a string further compact (stack up) to make solenoid (30nm fiber)
DNA Packing: Loops
- 100nm
- At base of loops is anchor points where topoisomerases are
- Loops generated via structural maintenance of chromosomes (SMC) complex
- Condensin protein is part of the SMC complex and functions as a motor protein to extrude DNA loops
- DNA translocating motor functions moves DNA at a rate of 1500bp/s
- Moves DNA relative to itself, condensins fixed
- Uses ATPase activity
- Condensin like proteins present in yeast, humans and bacteria
DNA Packing: Loops
DNA Packaging: Mitotic Chromosome
- Chromosome core structure is chromosome scaffold
- Loops going back and forth from scaffold
DNA Packaging: Mitotic Chromosome
- Interphase nucleus chromatin organisation
- Heterochromatin:
- Highly compact
- Generally inactive (eg genes of a particular cell type that is not that specific cell)
- Gene poor
- often located at periphery of nucleus
- Usually replicated in late S- not necessary
- Euchromatin:
- Less compact
- Gene rich
- Contains active genes
- Usually replicated in early S
DNA Packaging: Mitotic Chromosome
- How is compaction modulated?
- When DNA wraps around nucleosome core particles, histone tails stick out beyond the DNA
- This effects how nucleosomes interact with eachother (compaction) and other proteins eg TFs
- Histone tails are accessible to enzymes and can be chemically modified which change the properties of histone tails eg charge
- Allows for much variation and potential for complex information (epigenetic)= histone code
- Each cell type will have a different variation of its own histone code
- Common modifications: Acetylation, phosphorylation, methylation, ubiquitination
- Modifications usually occur to amino acids: arginine, lysine, serine
- Eg H3K27Me3- three methyl groups(M) to lysine (K) at amino acid position 27 in histone H3
- Reversible, pushes chromatin towards heterochromatin state, supressive
Histone tails stick out past DNA
DDR and Chromatin Modification
- The H2AX histone variant is part of the DDR
- It is incorporated all over the genome at low frequency
- Once near a ds break is gets phosphorylated by protein kinase on its serine and marks the location of the ds break to attract repair proteins
DDR + Chromatin Modification of Histones
- H2AX commonly used to visualise cells undergoing DDR
- UV light creates stripe of damage
- H2AX phosphorylated locally at damage
- Phosphorylated H2AX helps attract repair factors eg BRCA1 which is involved in recruiting other proteins to repair ds break
- Also recruits cohesin which fucntions during mitosis to keep chromatids together- helps prevent chromatids from separating to promote HR repair
Another Role of Histone Modifications
- Modulation of transcription
- There are many readers with different specificity that bind to histone tails
- Each modification of histone tails causes the binding of these
- Readers recruit or stabilizes other proteins, influencing transcription, recombination, replication and repair
- Influence transcription by determining chromatin accessibility to TFs
- Readers are released at mitosis by protein phosphorylation
Copying Epigenetic Info: reader-writer
- Each cell cycle has an opportunity for change (differentiation)
- Mostly the histone code is maintained in daughter cells, receive the same epigenetic profile (pattern of modifications of histones), unless differentiates
- Modified histones are shared in daughter cells so that half of the daughter nucleosomes recieve parental modified histones
- Reader-writer complexes re-establish the histone parental pattern
- They recognise the same modifications that they catalyse, bind the modifications on the daughter nucleosomes and recapitulate the modifications on others
Copying Epigenetic Info: reader-writer
DNA Methylation: Epigenetic Info
- DNA methylation is a type of DNA modification by DNA transferase (DNMTs) at CG dinucleotides (CpG)
- Promoters have CpG islands (runs of CpGs), methylation at these islands is reversible
- CpGs and their metyhlation have to be copied into daughter cells
- Upon replication half goes into daughter molecules
- CpG islands have symmetry (palindromic sequence) so if a pair retains methylation on cytosine, recognised by enzyme and methylation added to the other strand= recapitulation of original pattern
DNA Methylation: Epigenetic Info
Nuclear Organisation: Epigenetic Info
- Spatial organisation of chromosomes in a nucleus
- Chromosomes have preferred locations relative to others and their position is heritable
- Genes on different chromosomes can be part of the same megacomplex and regulated together which is related to their proximity in the nucleus
- Can visualise whole chromosomes or specific genes via fluorescence in situ hydribisation
Nuclear Organisation: Epigenetic Info
- Sequence proximity can be measured using proximity ligation
- Intact nucleus, apply chemical agent that cross links genes within particular protein complexes they are part of
- Fragment whole genome- bits in close proximity are crosslinked
- Add flags to loose ends and ligate ends
- Fragmentation to break up the bound parts and the flagged parts are isolated and sequenced
- Maps the frequency of the interaction of regions within a chromosome that are frequently associated with one another and regions associated with other chromosomes - not random
Proximity Ligation
Key Points
- DNA packaging is not uniform in the nucleus or in the cell cycle
- Nucleosomes are the basic unit of DNA packaging
- DNA modification (methylation) adds a layer of heritable information
- Complex patterns of histone modification enable or restrict access to other factors
- Epigenetic information is read and copied by dedicated enzymes
- Chromosomes and genes display characteristic spatial arrangements in the nucleus