NUCLEOSOME AND CHROMATIN

Aswathi K S1ST MSc BCMRoll no: 04

Central University of Kerala

Why DNA needs to be packed?1. Enormous length of genome. Human genome- 23 pair of

chromosome, Average length 1.3x10ˆ8 bp, aprox.

43mm For this amount of DNA to fit into

nucleus of size approx. 10-20µm require condensation of DNA

2. Structural complexity Association of DNA with a number of

proteins

Packing of DNA in eukaryotes

DNA almost 10cm long is converted to chromatin fibers 10-13nm in length and found dispersed in nucleus

At time of cell division these fibers condense into much larger and compact structure called chromosome.

Proteins important in maintaining chromatin structure are histones

Histones are small proteins containing lysine and arginine giving them a strong positive charge

Binding of negatively charged DNA to positively charged histones is stabilized by ionic bonds.

Mass of histones in chromatin approx = mass of DNA

Types of HistonesDivided to 5 main types1. H12. H2A 3. H2B4. H35. H4 Chromatin contains equal numbers of H2A,

H2B, H3 and H4 and about half that number of H1 molecules. Chromatin also contains diverse group of non histone proteins that play a variety of enzymatic, structural and regulatory roles.

NUCLEOSOME Basic unit of chromatin structure. Folding of DNA of enormous length to a nucleus

not more than 5-10µm achieved. First insight to folding: in late 1960s- X-ray

diffraction studies by Maurice Wilkins revealed that purified chromatin strands have a repeating structural subunit seen neither in DNA nor in histones alone.

Wilkins concluded that histones impose a repeating structural organisation upon DNA.

In 1974, Ada Olins and Donald Olins published electron micrographs of chromatin fibres isolated by avoiding harsh solvents

Chromatin fibers appeared as a series of tiny particles attached to one another by tiny filaments : ‘beads on a string’

This appearance lead to a suggestion that beads consist of protein and thin filaments connected to beads correspond to DNA.

We now reffer to each bead with its associated stretch of DNA as Nucleosome

Independent evidence for existence of repeating structure in chromatin was reported by Dean Hewish and Leigh Burgoyne – discovered that rat liver nuclei contain a nuclease that is capable of cleaving DNA in chromatin fibers.

DNA was exposed to its nucleases and the partially degraded DNA was purified to remove chromatin proteins

Upon electrophoresis found a distinctive pattern of fragments in which the smallest piece of DNA is of 200bp in length.

Nuclease digestion of DNA free protein does not give this fragment pattern

So they concluded that:1. Chromatin proteins are clustered along

the DNA molecule in a regular pattern that repeats every 200bp.

2. DNA located between these protein cluster is susceptible to nuclease digestion yielding fragments that are multiples of 200bp in length

To find out if the protein cluster postulated to occur at 200bp intervals correspond to the spherical structure on electron micrograph further analysis was done.

Chromatin exposed to micrococcal nuclease (like rat nuclease) and fragmented chromatin separated to fractions of varying size by centrifugation and observed by electron microscopy.

Smallest fraction found to contain single spherical particle, next fraction containing two clustered units and so forth.

When DNA analyzed by electrophoresis, DNA from fraction containing single particle measured 200bp, those containing 2 particles measured 400bp and so on

Therefore concluded that, the spherical particle in electron micrograph are associated with 200bbp of DNA.

This basic repeating unit containing an average of 200bp of DNA associated with protein particle is the Nucleosome

HISTONES First insight to molecular architecture of nucleosome emerged from the work of Roger Konberg, who was awarded Nobel Prize in 2006 for the series of

fundamental discoveries concerning DNA packaging and transcription in prokaryotes.

Konberg and colleagues showed that chromatin fibers composed of nucleosomes can be generated by combining purified DNA with a mixture of histones

When attempted to use individually purified histone, discovered that nucleosome could only be be formed when histone isolated gently.

H2A bound to H2B and H3 bound to H4. H2A-H2B and H3-H4 complex when mixed with

DNA, reconstituted chromatin fibers exhibiting nucleosomes.

Kornberg concluded : ‘H3-H4 and H2A-H2B complex are integral part of nucleosome’.

To understand nature of histone interaction, Kornberg and Jean Thomas treated isolated chromatin with a reagent that forms covalent crosslinks between protein molecule located close to eachother.

After crosslinking, protein isolated and analyzed by PAGE.

Protein complexes the size of 8 histones were prominent in such gels, suggesting that nucleosomal particle contains octamer of histones.

Given that: H2A-H2B and H3-H4 form tight complexes and these 4 histones are present in roughly equivalent amounts in chromatin, Kornberg and Thomas proposed that histone octamers are created by joining together 2 H2A-H2B dimers and 2 H3-H4 dimers , and Dna helix is wrapped around it.

In previous model, significance of H1 histone was not given.

H1 is not part of octamer. Nucleosomes are isolated by briefly

digesting chromatin with micrococcal nuclease and degraded until the 200bp DNA reaches the length of 146bp.

During final stage, H1 is released. Remaining particle: histone octamer

with 146bp DNA is called “core particle” and the DNA fragment degraded out is called “linker DNA” because it joins one nucleosome to the next

Chromatin Nucleosomes are packed to form

chromatin fibers and chromosome.

Thicker chromatin(10nm) are called chromatin fiber (30nm).

H1 facilitates packing into thicker chromatin fiber.

Next level of packing is the folding of 30nm fiber into looped domains. (ave 50,000 to 100,000 bp)

This arrangement maintained by attachment of DNA to insoluble network of non histone proteins, that form chromosome scaffold, to which long loops of DNA are attached.

Active DNA more tightly packed than inactive DNA, providing easier acess to proteins involved in transcription.

Tightly compacted chromatin: Heterochromatin – appear as dark spots in micrographs

Loosely packed, diffuse form of chromatin: Euchromatin

Heterochromatin contain transcriptionally inactive DNA

Euchromatin is associated with DNA being actively transcribed.

Chromatin in metabolically active cell found as euchromatin, but on cell division become compacted to generate a group of chromosomes.

Each chromosomme consist of duplicated DNA units called chromatids.

DNA Packing Ratio Used to quantify extend of folding of

DNA molecule. Calculated by determining total

extended length of a DNA molecule and dividing it by length of chromatin fiber or chromosome.

Initial coiling of DNA around histone reduces the length by a factor of 7

Formation of 30nm fiber result in 6 fold condensation.

Packing ratio of 30nm fibre is therefore 7x6=42.

By further foldind and coiling, overall packing ratio of euchromatin is about 750.

For heterochromatin and chromosomes, ratio even higher.

At time of cell division, typical human chromosome measures 4-5nm in length, but contains a DNA molecule measuring about 75mm if completely extended.

Packing ratio of chromosome falls in the ratio of about 15,000 to 20,000.