ROJW - chromatin Flashcards
Eukaryotic DNA is packed in nucleosomal arrays:
To form chromatin, DNA is wrapped around with histones to form nucleosomes which are then linked to one another by linker DNA, forming a bead-on-a-string structure.
Nucleosome’s existence in chromatin is proved by treating chromatin with nuclease which will only be able to cleave DNA in the linker region, not the DNA bound to the nucleosome.
Partial digestion can also be performed to cleave some of the linker regions, resulting in distinct DNA fragments containing one or more repeated units (di, tri, tetra nucleosome, etc.)
Nucleosome subunits
- consist of 2 of each of the 4 core histones: H2A, H2B, H3, H4 (8 in total)
- All of the core histones contain a central ‘histone fold’
o A long alpha helix flanked by short alpha helices
o A dimerization motif – allows histones to bind together in a specific way
2 H3-H4 form dimers that interact to form tetramers
2 H2A-H2B form dimers that interact to form tetramers
2 tetramers come together to form the histone octamer with a compact core and flexible tails
Histone Evolutionary Origin
- Histones are very highly conserved across the entire eukaryotic range
- Archaea has developed along eukaryotes for some time before branching off to its own domain
- Therefore, Euryarchaea also contain histones
o This suggests that histones emerged very early during evolution (more than 2 x billion [i.e. 2 x 109] years ago and have remained mostly unchanged since then
o Most of these euryarchaeota are hyperthermophiles (i.e. they live in very hot [80-120oC] environments, such as vulcanic thermal vents on ocean floors).
o It is therefore likely that histones originally evolved to stabilize and protect DNA under such extreme conditions
There is structural conservation between
Archaeal and Eukaryotic Histones
Histone-DNA interactions
- There are 5 distinct types of non-covalent interactions between histone & DNA:
o Hydrogen bonds
o Ionic (electrostatic) interactions
DNA is highly – charged & surface of histone octamer has + charged amino acids so they attract
o Nonpolar (hydrophobic) contacts
o Intercalation of arginines to contact phosphate backbone across the groove: ideally requires A-T base pairs
- The strength of these interactions is required to bend the 146 bp long DNA into two tight circles around the histones (which is normally energetically highly unfavorable because of the rigidity of double-stranded DNA!)
Upon interacting with histones, the DNA is still a double helix…
but the major and minor grooves of DNA become distorted during nucleosomal packaging
- Explains why:
o Endonucleases cannot cleave DNA when packed with nucleosome
o Transcription factors cannot bind to DNA when packed with nucleosome because their DNA binding domain was designed to bind regular B form DNA
what is required to form a more compacted chromatin structure
linker histones:
- such as H1and H5 that stabilize interaction between nucleosomes
- linker histones organize the entry and exit points of DNA on the nucleosome
o by binding across the top of nucleosome, constraining the way which DNA comes in/ go out of the nucleosome - extends nuclease protection to 168 - 200 bp
- The H1 linkers can then come together to form a higher order structure which is the 30 nm solenoid structure of chromatin fibres
- This higher order is more tightly packed – forms a repressive chromatin structure that will not allow high level of gene expression
- V.S. bead-on-a-string structure that is more permissive and allow gene expression to occur
ChromEMT
Recent research casts doubt on the relevance of the solenoid structure under in vivo conditions
ChromEMT technique was performed to study the chromatin structure
* Combines electron microscopy tomography (EMT) with a labelling method (ChromEM) that specifically stains DNA for electron microscopic observation
* This enables the ultrastructure of individual chromatin chains, heterochromatin domains, and mitotic chromosomes to be resolved
* Obtain serial optical slices that can then be reconstituted into detailed 3D models (build 3D chromatin structure in nucleus from stacks of images)
Results from ChromEMT:
- Little evidence for regular helical structures as predicted from solenoid model
- The chromatin chains are flexible and can bend at various lengths to achieve different levels of compaction and high packing densities (form more irregular structures, don’t take up higher order structures)
- Nucleosomes assemble into disordered chains with diameters between 5 and 24 nm. These chains display a variety of different structural conformations, particle arrangements, and packaging densities
- Therefore, solenoid model = not relevant anymore
Histone N-termini
- In addition to the conserved histone fold, histones also contain additional sequences, mostly at the N-termini, that play an important role in controlling the gene-regulatory properties of nucleosomes
- These additional sequences are highly flexible and therefore do not show up in X-ray crystal structures
- When histones form nucleosomes w/ DNA, the flexible N-termini emerge from the nucleosome and can stretch quite far out if fully extended! (but in vivo = coiled up in random conformation so not as extended)
N-terminal tails are important for:
site of post-translational modifications
- Acetylation & methylation of conserved lysine residues
o The acetylation & methylation patterns of N-termini of histones H4 and H3 are absolutely conserved in evolution (yeast to humans)
Process of acetylation
- Histone Acetyl-Transferase (HAT) eliminates the CoA group from Acetyl-CoA and transfers it on to the + charged amino group of lysine, so the lysine no longer has a + charge (removes lysine + charge)
o This + charge was important for histone binding to DNA
o Therefore, reduction of + charge causes histones to bind DNA less tightly due to less electrostatic interactions
o Allows for more permissive conformation of chromatin – open chromatin which is more accessible to transcription factors - Histone Deacetylase (HDAC) cleaves off acetyl group from lysine and restores the + charge amino group
o Restores more tightly packed chromatin structure closed chromatin – less accessible to TF, less transcribed/ switched off genes
Opening/closing of chromatin by gene-specific transcription factors
- Pioneer transcription factors are the only TF that can bind DNA during its packaging with histone in the form of nucleosomes
- Once these factors have bound, they can
o Recruit HAT to create a transcriptionally active chromatin environment or
o Recruit HDAC to create a repressed chromatin environment
Ex of yeast transcription factors: GCN4 and Ume6
GCN4 (Gene-specific activators – pioneer TF)
recruit GCN5 (histone acetyl transferases - HATs) locally via its activation domain to create a loose, hyperacetylated chromatin environment capable of initiating high levels of transcription initiation.
Ume6 (Gene-specific repressors – pioneer TF)
(Gene-specific repressors – pioneer TF) recruit Sin3 (histone deacetylases- HDACs) locally via its repressive domain to create a hypoacetylated chromatin environment which is inaccessible to the transcriptional machinery
Organization of chromatin
- Transcribed parts of the genome are present in the form of euchromatin (can be switched on and off by modifications)
o Nucleosomes are acetylated, low levels of linker histones - Many parts of the genome (mostly repetitive DNA surrounding centromeres) are permanently densely packaged in the form of heterochromatin
o Nucleosomes are methylated and contain special proteins (including linker histone H1) to maintain high packing density
