IT1: How is DNA packaged in the cell? Flashcards
How could Y chromosomes have degenerated?
Gain of a sex determining allele on one pair of equivalent autosomes that selected for male beneficial/female antagonist mutations on the chromosome. It would then be advantageous to retain this allele, so recombination suppression over all or most of that chromosome is selected for. The absence of recombination results in the accumulation of deleterious mutations, caused by Muller’s Ratchet, leading to degeneration of the chromosome.
Purifying selection also cannot occur, and so there’s an accumulation of transposable elements.
What is a nucleoid?
Meaning nucleus-like, it contains the genetic material of prokaryotic cells.
What is the structure of B-form DNA?
How does under-winding or over-winding impact the structure?
~10.5 bp per turn
Right-handed double helix
Under-winding: negative supercoils (right-handed)
Over-winding: positive supercoils (left-handed)
What leads to nucleoid formation in prokaryotes?
- Formation of negative supercoils by topoisomerases, such as DNA gyrase.
- Histone and nucleoid-associated proteins that organize the DNA into a compact structure that’s more resistant to damage.
What is a plectoneme?
A structural feature of super-coiled DNA, forming a twisted structure that resembles a series of loops.
[Think of when you over twist some rope and it forms individual loops coming off the central strand.]
Which nucleoid-associated proteins function to bridge and loop DNA?
Describe the structure and function of each.
Histone-like nucleoid structuring proteins:
- short protein
- binds AT-rich DNA via C-terminus
- N-terminus contains self-associating domains to form ‘daisy chain’ filaments which stiffen the DNA
SMC:
- 2 SMC monomers and a Kleisin subunit form a ring
- The ring captures DNA to enable looping
- Head domains have ATPase activity to pull DNA through the ring
Which nucleoid-associated proteins function to bend DNA?
Describe the structure and function of each.
Factor for inversion stimulation (FIS):
- Dimer
- Binds major groove of DNA
- Bends DNA between 50-90 degrees
Integration host factor (IHF):
- Heterodimer of alpha and beta subunits
- Prefers to bind AT-rich DNA
- Beta-ribbon arms protrude into the minor groove to kink the DNA 160 degrees.
Which nucleoid-associated proteins have functions beyond packaging DNA? What are these functions?
IHF can bend the DNA upstream of promoters to allow DNA elements bound by TFs get closer to the gene promoter - GENE REGULATION.
SMC complexes are loaded onto newly replicating daughter chromosomes to support chromosomes individualization and segregation into daughter cells - CHROMOSOME SEGREGATION.
How was chromatin discovered? What is its structure?
The term “chromatin” was first coined by German anatomist Walther Flemming in 1880, who observed it under a microscope while studying cell division in salamander larvae. Flemming noticed that the nucleus of the cell was stained differently from the surrounding cytoplasm, and he named the material that made up the nucleus “chromatin,” from the Greek word “chroma,” meaning color.
Negative stains showed a ‘beads on a string’ configuration, with particles of ~70A that had 15A gaps between them.
What experiment confirmed the presence of histones in chromatin?
Liver cells were treated with DNase before the DNA was isolated and separated on a gel. The gels showed a consistent band size of ~200bp at a time, and as the digestion went on for longer, the smaller species became more intense.
If the DNA was purified before DNase was added, this patterning wasn’t observed. This suggests that there’s some repetitive spacing of sites that are accessible to DNA, but would normally be protected.
Other work on purified DNA showed the presence of 5 distinct histone proteins, and that these formed the basic scaffold for chromatin assembly.
What was Roger Kornberg’s 1974 model of chromatin? How was this work tested?
- Chromatin is composed of a repeating unit of 2 of: H3, H4, H2A and H2B, and 200bp of DNA.
- Chromatin fibres consist of many of these units to form a flexibly jointed chain.
EM indicated a native chromatin configuration could only form when all of these parts were present, and adding H1 compacted the structure further. This lead to the coining of the term ‘nucleosome’ to describe the DNA/histone particles.
What did the atomic structure of the nucleosome reveal about its structure? Explain for the low-resolution, 3.1A, and 2.8A resolution structures that were solved.
LOW RESOLUTION:
There are ~1 3/4 turns of a left-handed coil of double-stranded DNA around a disk shape histone octamer.
The nucleosome has a dyad axis of rotational symmetry.
A further 3.1A structure was resolved to show histones are made up of 3 alpha helices that are linked by 2 short loops and have flexible N-terminal tails. H2A also has an extended C-terminal tail.
The 2.8A structure provided a detailed view of interactions where histone tails were positioned with respect to the DNA.
Describe the histone-fold structure and how this is used to form histone dimers.
The histone-fold structure consists of three alpha helices separated by two loops, and it is highly conserved across different histone proteins and species.
The skewed u-shaped folds of the histone monomers allows for them to slot together via a ‘handshake’ to form dimers.
How is DNA bent around the histone octamer? How is this stabilized?
DNA is bent around the histone at AT rich sequences through narrowing of the minor groove.
- It’s sequence-independent.
- Uses hydrogen bonds.
This is stabilized by the high-degree of positive charge in the histone octamer, coming from the >20% lysine and arginine residues.
How do prokaryotes and eukaryotes utilize DNA supercoiling to compact their DNA?
Prokaryotes use DNA gyrases to negatively supercoil their DNA, forming the nucleoid with plectonemes.
Eukaryotes use supercoiling to wrap their DNA around histone octamers and compact the DNA 6-7 fold.
What is the structure and role of histone H1 in the histone octamer?
Structure:
- Flexible N-terminus
- Intrinsically-disordered C-terminus that’s highly basic
- Lacks histone fold
- Globular domain sits on the dyad and interacts with linker DNA where it exits the nucleosome.
Function:
- Holds DNA in a more rigid and compact conformation
- Can be rapidly exchanging with other H1 molecules to alter chromatin structure of function, aiding in chromatin dynamics
How are new nucleosomes synthesized and deposited on DNA?
- HSP70/Hsc70/NASP promote folding and dimerization of H3/H4, whilst ASF1 binds H3 to prevent tetramerization.
- MCM2 shields DNA binding interface..
- Delivery to CAF-1 in the nucleus allows binding to the PCNA and supports tetramerization and deposition on the DNA.
- NAP1/FACT deposits H2A/H2B dimers on the tetrasome.
- Chromatin-remodelling enzymes wrap the rest of the DNA around the nucleosome.
H1 deposition is less well understood.
What is the chromatosome?
Nucleosome core particle + H1
How has the positioning of histones on DNA been studied? What did this reveal about histone occupancy across the genome.
EM images and digestion assays show homogeneous and uniform coverage of DNA…
Nucleosome mapping (e.g., DNase seq) showed nucleosome occupancy is high, with regular arrays that lack defined phasing. But some regions have more regular phasing with low or no occupancy at TSS.
The same can be said for other non-gene elements, such as enhancers, insulators and origins of replication.
What dictates nucleosome phasing and organization?
How was this shown?
- AT-rich sequence of DNA for nucleosome binding and bending. Shown via modeling predictions and testing these patterns.
[Other gene promoter sequences can also influence phasing.] - Chromatin-remodeling enzymes.
The models weren’t enough, but adding ATP to the patterns made them much more like in vivo patterns, suggesting ATP dependent processes…aka chromatin remodeling enzymes.
Deletions of the genes encoding these enzymes in yeast resulted in far less-defined nucleosome phasing. - DNA binding factors.
High occupancy of DNA binding factors competes with nucleosome occupancy, mostly at promoters, enhancers, insulators and origins. e.g., CTCF in mammals which binds insulators and causes high levels of phasing.
What are chromatin remodeling enzymes? Describe their structure.
Chromatin remodeling enzymes are a class of proteins that use the energy from ATP hydrolysis to alter the structure and position of nucleosomes on DNA.
There are several families of chromatin remodeling enzymes, each with their own unique structure and function.
- 2 conserved lobes for movement along the DNA
- ATPase domain + auxilliary domains
- ATPase domain powers movement (like 2 hands walking along the DNA)
How do remodeling enzymes move nucleosomes?
They use ATP to translocate or slide DNA over the surface of the nucleosome:
They anchor themselves to histones where the ATPase can then grab onto the DNA and translocate it towards the nucleosome dyad.
This process disrupts local histone-DNA interactions and as the DNA is pushed towards the dyad, the distortion is translated around the nucleosome to cause DNA translocation.
Why might defined phasing at certain regions of the genome be important?
TRANSCRIPTION/TRANSLATION:
Nucleosome occupancy can block access to the DNA.
Gene promoters have evolved features that create nucleosome-free regions which may be important for GTF and RNAPII binding.
How is phasing/occupancy of nucleosomes related to the function of the genome?
Nucleosome occupancy usually blocks transcription from occurring.
To overcome this, pioneer factors can recognize partial binding sequences on top of the nucleosome to recruit chromatin remodeling enzymes that displace the nucleosomes for transcription.
This is referred to as assisted loading and is likely key to how DNA sequences are unveiled for new functions.
What is the 30nm fiber, and how was it discovered?
The 30nm fiber is a compacted and more condensed form of chromatin that is formed when nucleosomes are further organized and packed together.
This was discovered using EM to visualize chromatin at higher resolution, demonstrating that increased cation charge partially shielded the DNA negative repulsion to create these higher order fibers.
What is the structure of the 30nm fiber?
The nucleosomes are stacked on top of each other in a zig-zag orientation with twisted tetranucleosome units.
H1 binds at the dyad and determine the trajectory of the exit or entry linker DNA.
How do H2A and H2B molecules interact in the 30nm fiber?
The interaction between H2A-H2B dimers in the 30nm fiber is thought to occur primarily through electrostatic interactions and hydrogen bonding between amino acid residues on the surface of the nucleosome.
This mediates stacking in the tetranucleosome unit.
What is the role of acidic patches in 30nm fiber formation?
The acidic patches on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome.
What experiment has suggested that the 30nm fiber doesn’t actually exist?
SAXS measurements on mitotic chromosomes from various cell types showed that it was ribosome contamination primarily causing the 30nm SAXS peaks.
When ribosomes were washed away, most cells showed no 30nm peak…
What is STORM, and how has it aided in determining chromatin organization in cells?
STORM is a super-resolution microscopy technique that uses fluorescent probes that can be turned on and off to obtain highly precise spatial information about the positions of individual fluorescent molecules in a sample.
STORM has been used to visualize the higher-order organization of chromatin, suggesting there’s no evidence for a regular 30nm fiber.
What is chromEM tomography, and how has it aided in determining chromatin organization in cells?
Combines cryo-EM with computational methods to generate 3D models of chromatin.
It showed that chromatin is far more heterogeneous than originally anticipated, with no evidence for a 30nm fiber.
Define the following terms:
- Euchromatin
- Heterochromatin
- Constitutive heterochromatin
- Facultative heterochromatin
Euchromatin: ‘open’ chromatin
Heterochromatin: ‘closed’ chromatin
Constitutive heterochromatin: constantly ‘closed’ chromatin
Facultative heterochromatin: silenced chromatin that can be activated when needed
Describe the genetic screens in Drosophila that identified Su(var) and E(var) genes, responsible for variegation and dose-dependence.
In these screens, researchers searched for mutations that caused changes in the expression of genes located near chromosomal regions known as “heterochromatin”. [Red vs white eye experiments]
Su(var) mutations suppressed the variegation phenotype that results from the abnormal expression of genes located near heterochromatin.
E(var) mutations enhanced the variegation phenotype in a dose-dependent manner, with the severity of the phenotype increasing with the number of copies of the mutation.
Su(var) and E(var) mutations were later discovered to impact the structure and organization of chromatin. Su(var) genes maintain condensed heterochromatin, whilst E(var) genes relax the structure.
Give an example of a protein encoded by a Su(var) gene. What is its function?
Su(var)3-9 encodes a histone methyltransferase that uses SAM to methylate K9 on H3 tails.
How do modifications to chromatin influence its function?
Give an example for each.
- Act as a binding site for reader proteins that then effect function.
E.g., Su(var)3-9 methylation promotes HP1 binding that dimerizes to hold the nucleosomes in a more compact configuration. - Directly influence nucleosome structure and interaction.
E.g., acetylation neutralizes the positive charge on histone lysines, reducing the thermostability of the nucleosome (if acetylated on the tail) and its affinity for DNA (if core is acetylated).
How does acetylation influence inter-nucleosome interactions?
The acidic patch on the face of one tetranucleosome unit interacts with the basic tail of H4 on the adjacent tetranucleosome.
Acetylation of that H4 tail prevents the interaction from occurring, resulting in a de-compaction that is similar to removing the H4 tail entirely.
What is TSA, and how has it been used to study the effects of acetylation on chromatin structure in vivo?
TSA inhibits HDAC activity and leads to histone hyperacetylation.
Super-resolution imaging shows a more uniform distribution of chromatin after TSA treatment.
What do bisulfite-seq and ChIP-seq reveal about chromatin structure?
Bisulfite seq:
Bisulfite treatment converts C to U, but leaves methylated C alone. NGS can then show the methylation status and identify CpG islands.
ChIP-seq:
Proteins (e.g., nucleosomes) are fixed on the DNA and immunoprecipitated to allow for identification of the regions of DNA to which they bind.
Where can DNA methylation be found in the genome, and what does it do?
Primarily found on CpG dinucleotides within vertebrate genomes. Short stretches of CpG islands escape DNA methylation (CpG islands) and these are normally associated with vertebrate gene promoters.
Active enhancers tend to be hypomethylated.
However, removal of DNA methylation does little to impact gene expression, suggesting it doesn’t primarily function to regulate gene expression. Instead, its removal can lead to increased expression of young retrotransposons. So it protects cells from retrotransposon elements jumping into coding elements, etc.
How are transposable elements suppressed in vertebrate genomes?
- General methylation (prevents TF binding and recruits HDACs to maintain compaction)
- SETDB1-H3K9me3 pathway (potentially recruits HP1 for compaction, or recruits the HUSH complex)
How does SETDB1 know to methylate parasitic DNA elements? How does this methylation then spread?
It relies on a family of KRAB domain zinc finger DNA binding proteins that recognize the sequences in transposable elements first. These proteins interact with an adaptor protein which recruits SETDB1 to nucleate H3K9me3.
KRAB DBPs are rapidly evolving, likely in a ‘red-queen’ fashion to keep up with the hundreds of new types of invasions.
SETDB1 forms an alternative complex with MMP8 that binds H3K9me3 to cause spreading of methylation.
What is the HUSH complex?
The HUSH complex consists of three core components: TASOR , MPP8*, and MORC2. These proteins are thought to work together to recruit additional factors that promote transcriptional repression of retroviral elements.
MORC2 is an ATPase that is thought to compact chromatin.
*MPP8 forms an alternative complex with SETDB1 to spread methylation on transposable elements.
