Organisation of Eukaryotic Chromosomes

ORGANISATION OF CHROMOSOMES
CHROMOSOME STRUCTURE
The human genome contains 3 x 109 bp. If the DNA of all 46 chromosomes from one cell was linked together, it would measure one meter in length. However, in human as well as other eukaryotes, genomic DNA can be highly folded, constrained, and compacted by histone and non-histone proteins into chromatin and chromosome. This is accomplished by proteins that successively coil and fold the DNA into higher and higher levels of organization.
Chromatin structure
Compacts DNA ~ 10,000 X
Fig. 8.22 The many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome
Copyright 2006 Pearson Prentice Hall, Inc.
PACKAGING OF CHROMOSOMES
Fig 2.7
1st Level of DNA Packing

The basic structural unit of chromatin, the nucleosome, was described by Roger Kornberg in 1974. Two types of experiments led to Kornbergs proposal of the nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with protein) yielded a continuous smear randomly sized fragments. These results suggest that the binding of proteins to DNA in chromatin protects the regions of DNA from nuclease digestion, so that enzyme can attack DNA only at sites separated by approximately 200 base pairs.
Electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs.
Level 1: Nucleosome - the most fundamental unit of packaging
Thus, both nuclease digestion and the electron microscopic studies suggest that chromatin is composed of repeating 200 base pair unit, which were called nucleosome.
individual nucleosomes = beads on a string
Nucleosomes: Basic Unit of Eucaryotic Chromosome Structure Comprised of both a Histone Protein Core and DNA
[A] Electron Micrograph of chromatin isolated from interphase [B] Chromatin that has been experimentally decondensed to visualize the nucleosomes or beads on a string.
Nucleosome
Reduces the length of a chromatin thread to about 1/3 its initial length. Nucleosome core particles contain 146 base pairs of DNA wrapped 1.7 times around a histone core consisting of two molecules each of H2A, H2B, H3, and H4 (the core histones/ histone octamer).
Fig. 8.18 Nucleosomes connected together by linker DNA and H1 histone to produce the beads-on-a-string extended form of chromatin
H1
Histone octomer
Linker DNA 10 nm chromatin is produced in the first level of packaging.

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
Eukaryotic chromosomal organization

2 main groups of proteins involved in folding/packaging eukaryotic chromosomes
Histones = positively charged proteins filled with amino acids lysine and arginine that bond Nonhistones = less positive

Histone proteins
Abundant Histone protein sequence is highly conserved among eukaryotesconserved function Provide the first level of packaging for the chromosome; compact the chromosome by a factor of approximately 7 DNA is wound around histone proteins to produce nucleosomes; stretch of unwound DNA between each nucleosome

Nonhistone proteins
Other proteins that are associated with the chromosomes Many different types in a cell; highly variable in cell types, organisms, and at different times in the same cell type Amount of nonhistone protein varies May have role in compaction or be involved in other functions requiring interaction with the DNA Many are acidic and negatively charged; bind to the histones; binding may be transient

Histone proteins
5 main types
H1attached to the nucleosome and involved in further compaction of the DNA (conversion of 10 nm chromatin to 30 nm chromatin) H2A Two copies in each nucleosome H2B histone octomer; DNA wraps H3 around this structure1.75 times H4
This structure produces 11nm chromatin
The nucleosome contains ~200 bp ofDNA associated with a histone octamer that consists of two copies each of H2A, H2B, H3, and H4. These are known as the core histones. The association of DNA with the histone octamer forms a core particle containing 146 bp of DNA
Core DNA has an invariant length of 146 bp, and is relatively resistant to digestion by nucleases. Linker DNA comprises the rest of the repeating unit. Its length varies from as little as 8 bp to as much as 114 bp per nucleosome. DNA is wrapped 1.65 times around the histone octamer.
11nm Fibre
Internucleosomal spacer (~ 60 bp linker DNA) One molecule of the fifth histone H1, is bound to the DNA as it enters and exists each nucleosome core particle. This forms a chromatin subunit known as chromatosome, which consist of 146 base pairs of DNA wrapped around histone core and held in place by H1 (a linker histone) Histone H1 (linker) binds and compacts nucleosomes to form a 11nm fibre which represents the 1st level of organisation.

Histone proteins
DNA is further compacted when the DNA nucleosomes associate with one another to produce 30 nm chromatin Mechanism of compaction is not understood, but H1 plays a role (if H1 is absent, then chromatin cannot be converted from 10 to 30 nm) DNA is condensed to 1/6th its unfolded size
Mechanisms to Form the 30 nm Fiber From Linear Nucleosomes

Zigzag model of compaction involves several mechanisms acting together. A larger histone, H1, acts to pull nucleosomes together and the histone tails may help to pull the nucleosomes together.
30 nm Fibre
Zig-zag stacking of nucleosomes then coiling results in a solenoid structure having 6 nucleosome per turn. A larger histone, H1, acts to pull nucleosomes together and the histone tails may help to pull the nucleosomes together. H1 molecules can interact with one another since they are present in the central core of the solenoid Packaging is 40 fold.
Fig. 8.20b Packaging of nucleosomes into the 30-nm chromatin fiber
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
Chromsome Scaffold/300 nm
Structural non-histone protein could be involved. The scaffold itself appears to contain several proteins, notably large amounts of histone H1 (located in the interior of the fiber) and topoisomerase II. The presence of topoisomerase II further emphasizes the relationship between DNA underwinding and chromatin structure.
SARs are very AT-rich fragments several hundred base

pairs in length that were first identified as DNA fragments that are retained by nuclear scaffold/matrix preparations. They define the bases of the DNA loops that become visible as a halo around extracted nuclei and that can be traced in suitable electron micro-graphs of histone-depleted metaphase chromosomes. They are possibly best described as being composed of
numerous clustered, irregularly spaced runs of As and Ts
Fig. 8.21 Model for the organization of 30-nm chromatin fiber into looped domains that are anchored to a nonhistone protein chromosome scaffold
MARs
DNA sites attached to proteinaceous structures in interphase nuclei are called MARS (matrix attachment regions); they are sometimes also called SAR (scaffold attachment regions). The nature of the structure in interphase cells to which they are connected is not clear. Chromatin often appears to be attached to a matrix, and there have been many suggestions that this attachment is necessary for transcription or replication. When nuclei are depleted of proteins, the DNA extrudes as loops from a residual proteinaceous structure.
A surprising feature is the lack of conservation of sequence in MAR fragments. They are usually -70% A-T-rich, but otherwise lack any consensus sequences. Other interesting sequences, however, often are in the DNA stretch containing the MAR. cis-acting sites that regulate transcription are common, and a recognition site for topoisomerase II is usually present in the MAR. It is therefore possible that a MAR serves more than one function by providing a site for attachment to the matrix and containing other sites at which topological changes in DNA are effected. The nuclear matrix and chromosome scaffold consist of different proteins, although there are some common components. Topoisomerase II is a prominent component of the chromosome scaffold, and is a constituent of the nuclear matrix. This suggests that the control of topology is important in both cases.

Compaction continues by forming looped domains from the 30 nm chromatin, which seems to compact the DNA to 300 nm chromatin Human chromosomes contain about 2000 looped domains 30 nm chromatin is looped and attached to a nonhistone protein scaffolding DNA in looped domains are attached to the nuclear matrix via DNA sequences called MARs (matrix attachment regions)

MARs are known to be near regions of the DNA that are actively expressed Loops are arranged so that the DNA condensation can be independently controlled for gene expression
Chromatin structure
300 nm fiber Loops of 30 nm fibers Attached to protein scaffold Attachment points correspond to boundary elements, isolating regions of differential gene expression Metaphase chromatin Coiling of the 300 nm fiber phosphorylation (especially of
histone H1) helps to pack nucleosomes together and thus tends to promote higher levels of chromatin compaction e.g. formation of metaphase chromosomes.
The Metaphase Chromosome

-formed by looping and coiling of condensed chromatin: assoc. with H1 phosphorylation
June 24, 2002
MBB 222/Lecture 20
46
Summary
2 nm DNA double helix
June 24, 2002
11 nm Nucleosome (11 nm fiber) Interphase chromatin: 30 nm 30 nm Fiber genes expressed in extended 300 nm Loops I loops (11nm fibers) Condensatio 700 nm Loops II n into metaphase chromosom 1400 nm chromosome es MBB 222/Lecture 20 48
Figure 8.30 Alberts, Mol. Biol. Cell.

Organisation of Eukaryotic Chromosomes

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How does DNA get packaged into chromatin and chromosomes?

How does DNA get packaged into chromatin and chromosomes?

What are the different levels of chromatin packaging?

What are the different levels of chromatin packaging?

ORGANISATION OF CHROMOSOMES

Copyright 2006 Pearson Prentice Hall, Inc.

1st Level of DNA Packing

Level 1: Nucleosome - the most fundamental unit of packaging

Linker DNA 10 nm chromatin is produced in the first level of packaging.

Eukaryotic chromosomal organization

Eukaryotic chromosomal organization

Eukaryotic chromosomal organization

Eukaryotic chromosomal organization

This structure produces 11nm chromatin

Eukaryotic chromosomal organization

Mechanisms to Form the 30 nm Fiber From Linear Nucleosomes

Fig. 8.20b Packaging of nucleosomes into the 30-nm chromatin fiber

SARs are very AT-rich fragments several hundred base

numerous clustered, irregularly spaced runs of As and Ts

Eukaryotic chromosomal organization

Eukaryotic chromosomal organization

The Metaphase Chromosome

June 24, 2002

2 nm DNA double helix

June 24, 2002

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