Nucleosome

Nucleosome (First level of chromatin organization)

Definition: Nucleosome is the fundamental repeating unit of eukaryotic chromosome, consisting of a length of DNA (200bp) coiled around a core of histones.

Nucleosome structure is produced through the interaction between DNA and histone proteins.

How it looks like?

A nucleosome consists of a protein core with DNA wound around its surface like thread around a spool.

Appearance of chromatin when viewed with electron microscope: Beads on string appearance

Figure 1: a) An electron micrograph of chromatin isolated from interphase nuclei showing its “beads on a string” character b) general representation of beads on a string appearance of chromatin.

Each nucleosome “bead” is a disc-shaped particle with a diameter of about 11 nm (110 A0).

Components in a nucleosome (200bp DNA + Histones)

Nucleosome = Nucleosome core particle + Linker DNA

The length of DNA that is associated with the nucleosome unit varies between species.

DNA in nucleosome can be divided into two regions = Core DNA (146 bp) + Linker DNA (~54 bp)

Linker DNA:

  • Linker DNA is double-stranded DNA in between two nucleosome cores that, in association with histone H1, holds the cores together.
  • Length varies from 10 – 80 bp depending on species and tissue type.
  • Linker DNA is seen as the string in the “beads and string model of chromatin structure.

Nucleosome core particle (NCP)

1) Histone proteins (form histone octamer): H2A, H2B, H3 and H4 (2 copies each)

2) Core DNA: (146 bp of DNA)

  • A 146bp segment of DNA wraps around the histone octamer 1.75 times (less than two turns) in left handed super helical fashion.

Since one turn around the nucleosome takes ~80 bp of DNA, two points separated by 80 bp in the free double helix are brought into close proximity on the nucleosome surface,

  • Thus, two sequences that are far apart can interact with the same regulatory protein to control gene expression.
  • The histones protect the segment of DNA in the nucleosome core from cleavage by endonuclease.
Figure 2: sequence of the DNA that lies on different turns around the nucleosome brought close together

Nucleosome parameters (figure 3)

  • The shape of the nucleosome corresponds to a flat disk or cylinder of diameter 11 nm.
  • The height of the cylinder is 6 nm, of which 4 nm is occupied by the two turns of DNA (each of diameter2 nm).
Figure 3: Nucleosome parameters

 

Figure 4: Structure of a) Nucleosome core particle b) Nucleosome
  • Each histone has an amino-terminal tail that extends out from the core structure.
  • These tails are flexible and contain a number of lysine and arginine residues.
  • Covalent modifications of these tails play an essential role in modulating the affinity of the histones for DNA and other properties.

Formation of NCP

  • The eight histones in the core are arranged into a (H3)2. (H4)2 tetramer and a pair of H2A.H2B dimers.
  • The tetramer and dimers come together to form histone octamer around which 146 bp of DNA wraps in a left- handed superhelical manner.

Chromatosome = Nucleosome core particle + H1 histone+ Linker DNA (~20 bp)

How chromatosomes are formed?

  • A chromatosome is formed as a result of histone H1 binding to a nucleosome.
  • The nucleosome core particle interacts with one molecule of histone H1 to form chromatosome containing ~166bp of dsDNA.
  • When H1 histone binds to the nucleosome, it wraps another 20 bp of DNA. As a result DNA makes two complete left handed super helical turns around the octamer and protects 166 bp of DNA from nuclease attack.
Figure 5: Structure of chromatosome

H1 histone

  • H1 is not a part of the nucleosome core particle but plays an important role in the nucleosome structure. (The H1 can be easily removed from chromatin without affecting the structure of the nucleosome, which suggests that its location is external to the nucleosome core particle).
  • The precise location of H1 with respect to the core particle is still uncertain. It binds to the linker DNA (The entry and exit site of linker DNA on the surface of the nucleosome core particle) in close proximity to nucleosome core.
  • H1 can stabilize both nucleosome structure and higher-order chromatin architecture
  • H1 helps to lock the wrapped DNA into place, acting as a clamp around the nucleosome octamer.
  • H1 histone attaches to the DNA near the nucleosome when a 10nm chromatin fiber undergoes the next level of packing.
  • Each chromatosome contain single molecule of H1 histone.
  • Histone H1 and its other variants are referred to as linker histones.
  • H1 histone is absent in Sacharomyces cerevisiae.
Figure 6: Comparison of structure of nucleosome core particle, chromatosome and nucleosome

Discovery of Basic repeating unit of chromatin (Nucleosome)

  • In 1974, Roger Kornberg proposed that chromatin is made up of repeating units, each containing 200 bp of DNA and histone octamer. These repeating units are known as nucleosome
  • Discovered by chemically digesting cellular nuclei and stripping away as much of the outer protein packaging from the DNA as possible.
  • When chromatin is isolated from the nucleus of a cell and viewed with an electron microscope, it frequently looks like beads on a string.
  • Individual nucleosomes can be obtained by treating chromatin with the endonuclease enzyme.

Experiment

 Enzyme used: Micrococcal nuclease (MNases) which cuts the DNA thread at the junction between nucleosomes).

Nucleases are a class of enzymes which will cause fragmentation of DNA at those sites which are not protected by proteins like histones.

  • If a small amount of nuclease is added to chromatin mixture (partial digestion), the enzyme cleaves the string between the beads, leaving individual beads attached to about 200 bp of DNA.
  • If more nuclease is added (more extensive digestion), the enzyme chews up the entire DNA between the beads and leaves a core of proteins attached to a fragment of DNA with 146 bp (NCP).

(DNA portions of nucleosome core particles are less accessible for DNAase)

  • When DNA from MNase-treated chromatin is electrophoresed on an agarose gel, a number of bands will appear.
  • By analyzing the sizes of each fragment produced, they could find that each fragment having a length that is a multiple (two, three, four times, etc.) of the size of mononucleosomal DNA (smallest unit).
Figure 7

Packaging ratio calculation (From linear dsDNA to nucleosome)

In nucleosome, each nucleosome core is wrapped around by 200 bp of DNA

1 bp = 0.3 nm

Length of 200 bp = 200 x 0.3 = 60nm

Diameter of nucleosome core = 11nm

Packaging ratio = 60/11 = ~6

This means that the DNA is compacted by a factor of 6 when linear dsDNA packed into nucleosomes

 

 

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