1. DNA Packaging

2. Lecture Overview
Structure of chromosomes and chromatin.
DNA packing and histone modification.

3. Objectives:
Understand the nature of chromatin - how DNA and histones interact to form nucleosomes.
Know the basic structures of the 10 nm chromatin fiber and the 30 nm chromatin fiber.
Realize that chromosomes are single pieces of DNA packaged into chromatin.

4. Genetic Material in the Living Cells
Cells contain a nucleus surrounded by a nuclear membrane in eukaryotic cells, and a nuclear region in the prokaryotic cells.
In a non-dividing cell the nucleus is filled with a thread-like material known as "chromatin".
Chromatin is made up of DNA and proteins (mainly histones and some non-histone acidic proteins).

5. The Normal Human Chromosomes
Normal human cells contain 23 pairs of homologous chromosomes:
22 pairs of autosomes.
1 pair of sex chromosomes.
Autosomes are the same in males and females
Sex chromosomes are:
XX in females
XY in males.
Both X are homologous. Y is much smaller than X and has only a few genes.

6. One member of each chromosome pair is derived from each parent.
Chromosomes
One member of each chromosome pair is derived from each parent.
Somatic cells have diploid complement of chromosomes i.e. 46.
Germ cells (Gametes: sperm and ova) have haploid complement i.e 23.
Individual chromsomes are recognized by
arm lengths ( p- short, q -long).
centromere position (metacentric, sub-metacentric, acrocentric, telocentric).

7. Composition of Chromosome
DNA
Histones
(Major proteins) q
Non-histone
(Small amounts)
The chromosomes themselves are macromolecular entities that must be synthesized, packaged, protected, and properly distributed to daughter cells at cell division.
Significant segments of every chromosome are dedicated to these functions.

8. DNA Condensation: Why?

9. A severe problem of packaging
Largest human chromosome: ~3 x 108 bp
A typical cell = 10 μm = 10 x 10-6 m
Therefore the DNA must be compacted ~104-fold.
This is like fitting an 11-mile-long string into a 6-foot box
How long is it?

10. DNA packing- Superstructure
Higher-order DNA compaction in a eukaryotic chromosome.  This model shows the levels of organization that could provide the observed degree of DNA compaction in the chromosomes of eukaryotes. First the DNA is wrapped around histone octamers, then H1 stimulates formation of the 30 nm filament. Further levels of organization are not well understood but seem to involve further coiling and loops in the form of rosettes, which also coil into thicker structures. Overall, progressive levels of organization take the form of coils upon coils upon coils. It should be noted that in cells, the higher-order structures (above the 30 nm filament) are unlikely to be as uniform as depicted here.

11. Eukaryotes contain thousands of times more DNA than do bacteria, and as a result, the DNA–condensation problems of eukaryotes—compacting the DNA so that it fits in the cell nucleus—are more complex than those of bacteria.
Bacteria do not contain nucleosomes, although they have small, basic (positively charged) proteins that are involved in condensing their DNA.

12. Continue..
DNA compaction must be dynamic, because changes in the degree of condensation must occur quickly and when needed, as the cell passes through the stages of the cell cycle.
Furthermore, when in its most highly compacted form, DNA is not accessible to transcription or replication enzymes, so it must be able to rapidly expose regions containing genes that are required at any given moment, and then condense again.
Modification enzymes that alter the state of DNA condensation, and can target their activity to specific regions of the chromosome that must be transcribed or replicated.

13. Nucleosomes: The Basic Units of DNA Condensation
The material of chromosomes, both protein and DNA, is often referred to as chromatin. The protein component is about equal in mass to the DNA component.
Histones constitute the largest protein component of chromatin, are highly conserved, basic proteins that assemble into octameric complexes containing two each of four different histone subunits.
DNA wraps around the histones to form condensed nucleosomes.

14. Nucleosome (10 nm diameter):
8 histones in bead & 1 outside.
Each bead: is surrounded by 140 bpDNA and there are 60 bp in the linker region.
Space between beads is about 14 nm.

15. Continue..
Histones are rich in the basic amino acids arginine and lysine, which together make up about 25% of the amino acid residues in any given histone protein.
Histone proteins are highly conserved among eukaryotic cells.
Histones H3 and H4 are nearly identical in all eukaryotes, suggesting strict conservation of their functions.
Histones H1, H2A, and H2B show less sequence similarity, but on the whole, they are more conserved than other types of proteins.
Salt bridges between positively charged histones and negatively charges DNA play a major role in stabilizing DNA-histone complex

16. Histone Octamers Organize DNA into Repeating Units
The first evidence that DNA is packaged into regularly organized units came from studies in which chromosomal DNA was treated with a nonspecific DNA nuclease, that cuts DNA wherever it is not associated with proteins.
The digested DNA fragments were then analyzed for size in an agarose gel.
If DNA is packaged by proteins into units of a particular size, the nuclease would cleave the DNA between these units, and the protected DNA segments would migrate in the gel as a ladder of unit-sized bands.
The results of such experiments revealed a series of regularly spaced DNA bands about 200 bp apart, indicating that DNA is packaged by proteins into units that encompass approximately 200 bp

17. The earliest evidence of DNA packaging.
Isolated chromatin was treated with nuclease and analyzed by agarose gel electrophoresis.
The result was a DNA ladder of fragments that differed in length by 200 bp, suggesting that DNA packaging involves a repeat unit of 200 bp.
[Source: Roger Kornberg, MRC Laboratory of Molecular Biology.]

18. Histones- main packing proteins.
Consist of 5 classes: H1, H2A, H2B, H3, H4.
When the protein-DNA units (nucleosomes) were examined by SDS−polyacrylamide gel electrophoresis (SDS-PAGE), four histone proteins (H2A, H2B, H3, and H4) were found in approximately equimolar ratios .
A fifth histone (H1) was present in about half the amount relative to the other four histones. H1 is Lys rich.
The five histones have molecular weights (Mr) between 11,000 and 21,000.
Histone representation in nucleosomes.
Histones within nucleosomes were separated by SDS–polyacrylamide gel electrophoresis.
Measurement of the band intensity showed histones H2A, H2B, H3, and H4 present in equal stoichiometry, and histone H1 at about half the level of the other histones. [Source: S. Panyim and R. Chalkley, Archiv. Biochem. Biophys. 130:337–346, 1969.]

19. Characteristics of Histones

20. Types of histone modifications
Chromatin Modifications
Functions Regulated
Acetylation
Transcription, Repair, Replication, Condensation
Methylation (lysines)
Transcription, Repair
Methylation (arginines)
Transcription
Phosphorylation
Transcription, Repair, Condensation
Ubiquitylation
Sumoylation
ADP ribosylation
Deimination
Proline Isomerization
Post-translational modifications on histone proteins alter chromatin structure and, consequently, chromatin function

21. Nucleosomes
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, and H4. These four histones have come to be known as the core histones.
The remainder of the DNA serves as a linker between nucleosomes, to which histone H1 binds.
Under physiological conditions, formation of the histone octamer from individual histone proteins requires the presence of DNA.
Nucleosomes as beads on a string. 
Regularly spaced nucleosomes consist of core histone proteins bound to DNA.

22. Histone H1 Binds the Nucleosome to Form the Chromatosome
The histone octamer and associated DNA that form the nucleosome combine with histone H1 to form the chromatosome.
The addition of H1 to a nucleosome results in protection of an additional 20 to 22 bp of linker DNA adjacent to the nucleosome, and thus H1 is often referred to as the linker histone.
Only one H1 subunit is present per chromatosome, unlike the core histones, which are present in two copies each.
DNA binding in H1 is intrinsic to the central globular region, which contains two DNA-binding sites.
H1 binds only one of the linker DNA strands, and the second DNA site in histone H1 binds to the central region of the DNA supercoil in the nucleosome
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.

23. Chromosomes Condense into a Compact Chromatin Filament
Nucleosomes condense into a compact filament with a width of about 30 nm, referred to as the 30 nm filament.
H1 promotes condensation into the 30 nm filament, but it is not essential for forming the filament.
In contrast, the N-terminal tails of the core histones are absolutely required, suggesting that the tails provide important nucleosome-nucleosome contacts needed for 30 nm filament formation.
There are two most widely accepted models for nucleosome arrangement in the 30 nm filament:
Solenoid model
Zigzag model

24. The 30 nm filament, a higher-order organization of nucleosomes.
The compact filament is formed by the tight packing of nucleosomes. Two proposed models of filament structure are (A) the solenoid model and (B) the zigzag model.
[Source: (a) Barbara Hamkalo, Department of Molecular Biology and Biochemistry University of California, Irvine.]

25. Higher-Order Chromosome Structure Involves Loops and Coils
Inside chromosomes, DNA is much more highly condensed than in the 30 nm filament.
30 nm filaments is appear to be organized in loops estimated at 40 to 100 kbp long.
chromosomal scaffold: Proteinaceous residue after extraction of histones from chromosomes, comprised mainly of Structural maintenance of chromosomes (SMC) proteins.
Regions of the DNA interact with chromosomal scaffold proteins to give a protein core with DNA loops sticking out of it.
This protein core then coils up to further package the DNA into the chromatids that are visible by light microscopy in metaphase.

26. Summary
Chromatin is the sum of the protein and nucleic acid that comprise the material of chromosomes.
The basic unit of DNA packaging is the nucleosome, composed of about 200 bp of DNA wrapped around a histone octamer.
The histone octamer contains a tetramer of histones H3-H4 and two dimers of histones H2A-H2B. The amino acid sequences of the histones are highly conserved among eukaryotes.
Nucleosomes are linked together by DNA with one bound molecule of H1.

27. Summary of Chromosome Folding
Level of folding
Consists of
Base pair/turn
DNA double helix
Nucleotides 10
Nucleosomes
200 bp each
100
30 Nanometer fiber
6 Nucleosomes /turn
1,200
Loops
50 Solenoids/loop
60,000
Miniband
18 loops
1,080,000
Chromatid
1,000,000 minibands