Chapter 10: Nucleosome: The Basic Units of DNA Condensation
How is chromosome structure regulated?
"open" chromatin structure = active gene expression "closed" chromatin state = repressed gene expression
Explain how bacterial DNA is organized
-Bacterial DNA is also highly organized -Bacteria lack nucleosomes but they do contain abundant histonelike proteins. -DNA is constrained in looped domains.
What are the functions of some histone variants
-H3.3 actively transcribe genes -H2AX involved in DNA repair, (phosphorylation attracts repair proteins) -H2AZ actively transcribe genes -CENPA associated with the repeated DNA sequences in centromeres and contains a large extension that connects to the kinetochore.
What are some of the Histone variants
-H3.3 and CENPA are H3 variants - H2AX, H2AZ and macroH2A are H2A variants
Explain the role of chaperones in nucleosome assembly
-Histone chaperones are required to assist the assembly of histone octamers onto DNA. -Histone chaperones are acidic proteins that bind either the H3-H4 tetramer or the H2A-H2B dimer. Chaperone mediated nucleosome assembly occurs in two steps 1) CAF-1 chaperone deposits and H3-H4 tetramer onto the DNA 2) NAP-1 chaperone assembles two H2A-H2B heterodimers with the H3-H4 heterotetramer to form the complete nucleosome
Explain the mechanism behind the histone code
1) The Gcn5 coactivator binds within a complex of proteins to a specific DNA sequence upstream of the gene. -Gcn5 subunit is an acetyltransferase (HAT) that acetylates lys8 of H4 and lys of H3.
What type of modifications of the N-terminal tails of histones plays a major role in altering chromatin structure
1) acetylation of lysine 2) methylation of lysine & arginine 3) phosphorylation of series 4) Ubiquitination of lysines
How many bp of DNA supercoil around the octamer?
146 bp of DNA supercoil around the octamer
What is the acetylation of lys residues performed by and what does it do?
Acetylation of Lys residues is performed by enzyme called histone acetyltransferases (HATs). Acetylation is associated with enhanced accessibility to DNA and consequent transcriptional activation. Deacytlation leads to transcriptional repression.
What does acetylation do to lysine?
Acetylation, which neutralizes the charge on lysine, simply relaxed the grip of a nucleosome on the DNA, thereby opening chromatin to regulatory proteins.
What are some of the functions of the chromatin remodeling complexes.
All chromatin remodeling complexes contain a conserved ATPase subunit and use the energy from ATP hydrolysis to disrupt the many contact between nucleosomes and DNA, allowing nucleosomes to be ejected or repositioned on the DNA.
Explain the experiment of repression of transcription by histones.
An in vitro transcription extract used to monitor transcription from a promoter in the presence or absence of histone proteins and the transcription activator Sp1. Adding core histone proteins results in decreased RNA synthesis. As histone H1 is titrated into the assay the transcription is diminished further. This suggests that H1 further compacts the chromatin and represses transcription.
Why do we need basic histone side chains?
Basic histone side chains are needed to neutralize the negative charge of the DNA's phosphodiester backbone. Charge neutralization is important in DNA condensation, especially in the further stages of compaction.
How can you determine the position of nucleosomes on genomic DNA
Chip-Seq and ChIP-Chip technique is used 1) Cells are treated with formaldehyde to covalently cross-link nucleosomes to DNA. 2) The cells are disrupted and genomic DNA is digested with micrococcal nuclease. 3) Antibodies to a histone is used to immunoprecipitate the nucleosome-DNA complex. Any DNA not bound to the histone is digested and washed away 4) Protein-DNA cross-links are broken and the released DNA is sequenced. 5) Released DNA is labeled and used to probe a microarray representing the genomic sequences 6) Pattern of hybridization on the microarray reveals the DNA sequences that associate with the nucleosomes. (peaks indicate regions of DNA bound to histones, and valleys indicate regions of DNA free of histones).
Define Chromatin and nucleosome
Chromatin : material that makes up the chromosome that contains protein and DNA Nucleosome: histones + DNA
What are two classes of proteins that alter nucleosome arrangements
Chromatin Remodeling Complex: slide the nucleosome to a different location or eject it from the DNA or replace it with a new nucleosome that contains a variant histone subunit. Histone Modifying Enzymes: covalently modify the N-terminal tails of histones, by attaching chemical groups to specific amino acid residues o nucleosome subunits. - Histone modifications are inheritable
Explain the histone-fold motif.
Each histone contains a histone-fold motif which are 3 alpha helices linked by two short loops.
Explain the folding of the histone fold dimer
Each histone fold dimer forms a V-shaped structure that contains three DNA-binding sites. The contacts between histones and DNA are mainly between the histone fold and the phosphodiester backbone in keeping with the non-specific binding of nucleosomes to DNA.
Explain the binding of DNA by histone H1
H1 has two DNA-binding sites through which it makes contact with one arm of linker DNA and the central region of the DNA wrapped around the histone octamer. In an electromicrograph, H1 increases the zigzag appearance by decreasing the DNA entry/exit angles.
Give one example of a histone chaperone
HIRA is a chromatin remodeling complex but can also be considered as a histone chaperone helping to ensure the proper assembly and placement of nucleosome.
Where do histone octamers assemble particularly well ?
Histone octamers assemble particularly well with sequences where two or more A=T bp are staggered at 10 bp intervals because DNA is naturally bent at these sequences, and when two or more consecutive A=T bp are spaced along the same face of the helix, the DNA bends into a circle.
Explain the histone representation in nucleosome?
Histones H2A, H2B, H3, H4 present in equal stoichiometry. The five histones have molecular weights between 11,000- 21,000. Histones are rich in the basic amino acids arginine and lysine.
What happens to the histone in the absence of DNA
Histones form an octamer structure but under physiological conditions this only occurs when DNA is present. In the absence of DNA, the highly conserved H3 and H4 subunits form a tightly associated heterotetramer that contains two of each subunit, and the less conserved H2A and H2B subunits form form a heterodimer.
Define epigentic inheritance
Inheritance of genetic properties that are not encoded in the DNA sequence.
Where did the first evidence that DNA is packaged into regularly organized units come from?
It came from studies in which chromosomal DNA was treated with a nonspecific DNA nuclease, such as micrococcal nuclease, that cuts the DNA wherever it is not associated with proteins. The results were analyzed by agarose gel electrophoresis which gave a DNA ladder of fragments that differed in length by 200 bp suggesting that DNA packaging involves a repeat unit of 200 bp.
What is the histone variant MacroH2A involved in?
It is involved in X chromosome inactivation, which shuts down one of the two X-chromosomes in the cells of female mammals.
What did Roger Kornberg proposed on how histones are organized within the nucleosome.
Kornberg suggested that most of the 200 bp of DNA in a protein-DNA unit is wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, H4. (core histones). The remainder of the DNA serves as a linker between nucleosomes to which histone H1 binds.
What are the two most widely accepted models for nucleosome arrangement in the 30nm filament fit
Solenoid model: the nucleosome array adopts a spiral shape, in which the flat sides of adjacent nucleosome disks are next to each other. Zigzag model: zigzag histone pairs stack on each other and twist about a central axis.
Describe the mechanism for chromatin remodeling complexes
Some chromatin remodeling complexes mobilize nucleosomes by forming a DNA loop within the nucleosome. Loop is propagated around the histone octamer, with the net effect that the nucleosome slides to a new segment of DNA. This leads to enhanced DNA accessibility (exposing promoter that was occluded by nucleosome). -The same mechanism can reposition to unexposed the promoter region and repress gene transcription. -Some chromatin remodeling complexes can also eject a histone octamer to generate a nucleosome free region for transcriptional activation.
Explain the significance of the Histone Tails
The N-termini of the histones protrude from the core particle and are less ordered. Histone tails do not contribute much strength to DNA binding but they form intermolecular contacts with adjacent nucleosome particles and organize nucleosomes into a higher-order chromatin structure. Histone tails regulate chromatin structure and are a target of numerous chemical modifications that control the access of regulatory proteins to DNA.
How can you move or push the nucleosome ?
The protein complexes contain an ATPase domain that releases chemical bond energy by rearranging the hydrogen bond. They could provide strain on the fibers that could pop out or release the nucleosome.
What happens if the internucleosome connections are tight.
The tighter the internucleosome connections mediated by the histone tails, the less accessible is the DNA to transcription factors and other proteins .
What is another way in which histone tail modification function?
They function by recruiting enzymes that recognize particular amino acid residues. Proteins with bromodomain motifs recognize acetylated lys residues and may help stabilize the open chromatin state Methylated histones are recognized by chromodomain proteins that may help promote the closed state.
Histones which consists of basic amino acids are highly conserved throughout eukaryotic evolution which suggests?
This suggest not only the strict conservation of their function but also a common ancestor.
What happens when a double strand break occurs?
When a double strand break occurs nearby molecules of H2AX become phosphorylated at Ser139 in the C-terminal region attracting DNA repair proteins.
What are the three main classes of chromatin remodeling complexes
chromatin remodeling complexes consists of 2 to 17 subunits and can be divided into three main classes: SWI/SNF (switch sniff), ISWI (Imitation switch), Mi2/NURD.
What would you see differently on the gel electrophoresis analyses if there were no regular repeating unit of protein-DNA packaging.
you would see protein would be distributed on DNA in a random way and nuclease digestion would produce a smear of DNA fragments with no regular pattern.