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Genomeorganization
In EukaryotesIn Eukaryotes
Arun Viswanathan
IInd Sem, M.Sc.BMB
© Arun Viswanathan
40 km wire in a tennis ball !
• Each cell has approximately 2meters of DNA
• Nucleus is only about 6µm in diameter
• In eu...
Genome organization
Chemical composition of chromatin
• DNA (20-40%)
most important chemical
constituent of chromatin
• RNA (05-10%)
associate...
Beads on a String
• Beads on a string structure is the primary level of DNA packaging
• They are often called as 11nm fibr...
Beads on a String
• Histone are highly basic (+ve charged),
• Rich in basic amino acids Arginine and Lysine
• Five Major c...
Histones
A 147bp segment of DNA then wraps around the histone octamer 1.65 times. Each
Nucleosome particle are separated f...
Nucleosomal Assembly
Histones are predominantly basic proteins but also contain hydrophobic and
acidic patches. They repel...
Histone-DNA interactions
1. Electrostatic Interactions:
Helix-dipoles form α
helixes in H2B, H3, and H4
cause a net +ve ch...
Histone tail, Histone code & Epigenetics
• There are eight N-terminal domain/Tail domain in histone core.
• These tail dom...
The 30nm fiber
• With the help of H1 the 11nm fiber compress to form
more compact 30nm fiber. H1 primarily is in contact w...
The 30nm fiber
•In the one-start solenoid
model, bent linker DNA
sequentially connects each
nucleosome cores, creating
a s...
The 30nm fiber
In the two-start zigzag
model, straight linker DNA
connects two opposing
nucleosome cores, creating
the opp...
‘One-start’ Helix
(Solenoid)
‘Two-start’ Helix
(ZigZag)
Intermediate 30 nm fibers
Four proposed structures of the 30 nm chromatin filament for DNA
repeat length per nucleosomes r...
Higher chromatin organizations
(Metaphase Chromosome)
• We know very less about higher chromosomal
levels of genome organi...
Higher chromatin organizations
(Metaphase Chromosome)
Higher chromatin organizations
(Metaphase Chromosome)
Higher chromatin organizations
(Metaphase Chromosome)
Higher chromatin organizations
(Interphase Chromosome)
• Determining how the Interphase chromosome is packed
was a great d...
Higher chromatin organizations
(Interphase Chromosome)
• The key experiment to
distinguish between two
models was carried ...
• During interphase, each chromosome occupies a spatially limited, roughly
elliptical domain which is known as a chromosom...
Chromosome Territory (CT)
Recurrent Clusters
A) Chromosome territories (green) in liver cell nuclei (blue). B) Visualizati...
Chromosome Territory (CT)
• Large areas of chromosomal identity between different
species that have been maintained throug...
Chromosome Territory (CT)
Movement of CT
GENE OFF GENE ON
Chromosome Territory (CT)
FISH of Human interphase nucleus
10µm
Other domains in nucleus
• Transcription factories
– transcription is spatially organized into discernable
nuclear structu...
Molecular Models of looping
• Random loop Model
oWith loops at all scales > 150bp
• Multi-loop model
oExplains 120kbp rose...
Sequential organization
Sequential organization
Tandem repeats
Microsatellite DNA
• Unit - 2-4 bp (most 2).
• Repeat - on the order of 10-
100 times.
• Location - General...
Interspersed Repetitive DNA
• Interspersed repetitive DNA accounts for 25–40 % of mammalian DNA.
• They are scattered rand...
Interspersed Repetitive DNA
• Retrotransposons are:
 long terminal repeat (LTR) Any transposon flanked by Long
Terminal R...
Gene rich regions have been
visualized with a fluorescent probe
that hybridizes to the Alu
interspersed repeat, which is
p...
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Genome organisation

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A detail ppt about Genome organization with focus on all levels of organization. Most recent research and findings about CT is also added in this ppt. Detail account of 30nm fiber and its ultra structure and types is also included.

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Genome organisation

  1. 1. Genomeorganization In EukaryotesIn Eukaryotes Arun Viswanathan IInd Sem, M.Sc.BMB © Arun Viswanathan
  2. 2. 40 km wire in a tennis ball ! • Each cell has approximately 2meters of DNA • Nucleus is only about 6µm in diameter • In eukaryotes DNA occurs as highly condensed form during cell division as Chromosomes. • 3.2x 109 nucleotides is packed into 24 different chromosomes • DNA is highly negative in charge. How it is possibly wind over another without repulsion? • How can genomic processes like replication and transcription is possible in such tightly winded structures? • the Disentanglement time for the transition from interphase to metaphase chromosomes of size 100 Mb is in the order of 500 years • Each cell has approximately 2meters of DNA • Nucleus is only about 6µm in diameter • In eukaryotes DNA occurs as highly condensed form during cell division as Chromosomes. • 3.2x 109 nucleotides is packed into 24 different chromosomes • DNA is highly negative in charge. How it is possibly wind over another without repulsion? • How can genomic processes like replication and transcription is possible in such tightly winded structures? • the Disentanglement time for the transition from interphase to metaphase chromosomes of size 100 Mb is in the order of 500 years
  3. 3. Genome organization
  4. 4. Chemical composition of chromatin • DNA (20-40%) most important chemical constituent of chromatin • RNA (05-10%) associated with chromatin as; rRNA, mRNA, tRNA • Proteins (55-60%) Histones: very basic proteins, constitute about 60% of total protein, almost 1:1 ratio with DNA. Five Types: H1, H2a, H2b, H3 and H4 • Non-Histones: They are 20% of total chromatin protein: • Nucleosomal Assembly Proteins (NAP), Other Histone chaperones Chromosome remodeling complexes • Structural (actin, L & B tubulin & myosin) contractile proteins, function during chromosome condensation & in movement of chromosomes. • all enzymes and cofactors – involved in replication, transcription and its regulation • DNA (20-40%) most important chemical constituent of chromatin • RNA (05-10%) associated with chromatin as; rRNA, mRNA, tRNA • Proteins (55-60%) Histones: very basic proteins, constitute about 60% of total protein, almost 1:1 ratio with DNA. Five Types: H1, H2a, H2b, H3 and H4 • Non-Histones: They are 20% of total chromatin protein: • Nucleosomal Assembly Proteins (NAP), Other Histone chaperones Chromosome remodeling complexes • Structural (actin, L & B tubulin & myosin) contractile proteins, function during chromosome condensation & in movement of chromosomes. • all enzymes and cofactors – involved in replication, transcription and its regulation
  5. 5. Beads on a String • Beads on a string structure is the primary level of DNA packaging • They are often called as 11nm fibre • The diameter of “beads” is 11nm • The beads are made of proteins called as Histones © Lehninger Principle of Biochemistry, Michael M.Cox, David L. Nelson, Fifth Edition W.H. Freeman And Company, New York
  6. 6. Beads on a String • Histone are highly basic (+ve charged), • Rich in basic amino acids Arginine and Lysine • Five Major class: H1, H2A, H2B, H3, H4 • Amino acid sequence of H3 and H4 are highly conserved • Histones and DNA along with NAP form a condensed structure called Nucleosome. It is the fundamental structural unit of chromatin. • The highly basic nature of Histones, aside from facilitating DNA Histone interactions, contributes to their water solubility. • H1 is present in half the amount of the other four histones. • Histone are highly basic (+ve charged), • Rich in basic amino acids Arginine and Lysine • Five Major class: H1, H2A, H2B, H3, H4 • Amino acid sequence of H3 and H4 are highly conserved • Histones and DNA along with NAP form a condensed structure called Nucleosome. It is the fundamental structural unit of chromatin. • The highly basic nature of Histones, aside from facilitating DNA Histone interactions, contributes to their water solubility. • H1 is present in half the amount of the other four histones. Content of basic amino acids Histones Molecular Weight Number of AA residue Lys % Arg % Total % H1 21,130 223 29.5 11.3 40.8 H2A 13,960 129 10.9 19.3 30.2 H2B 13,774 125 16 16.4 32.4 H3 15,273 135 19.6 13.3 32.9 H4 11,236 102 10.8 13.7 24.5
  7. 7. Histones A 147bp segment of DNA then wraps around the histone octamer 1.65 times. Each Nucleosome particle are separated from each other by a linker DNA, which can be of fewer nucleotides up to about 80. The term nucleosome refers to a nucleosome core particle plus an adjacent linker DNA. On an average, nucleosome repeat at intervals of about 200 nucleotides. A diploid Human cell contains about 30 million nucleotides !!
  8. 8. Nucleosomal Assembly Histones are predominantly basic proteins but also contain hydrophobic and acidic patches. They repel each other at physiological pH and form non- nucleosomal aggregates with DNA. Histone chaperones prevent these nonspecific interactions and can direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange.
  9. 9. Histone-DNA interactions 1. Electrostatic Interactions: Helix-dipoles form α helixes in H2B, H3, and H4 cause a net +ve charge to accumulate at the point of interaction with -vely charged phosphate groups on DNA 2. Hydrogen bonds: between the DNA backbone and the amide group on the main chain of Histone proteins 3. Non-polar interactions: between the Histones and sugars on DNA 4. Salt bridges and hydrogen bonds: between side chains of basic AA (especially lys and arg) & phosphate oxygens on DNA 5. Non-specific minor groove insertions: of the H3 and H2B N-terminal tails into two minor grooves each on the DNA molecule 1. Electrostatic Interactions: Helix-dipoles form α helixes in H2B, H3, and H4 cause a net +ve charge to accumulate at the point of interaction with -vely charged phosphate groups on DNA 2. Hydrogen bonds: between the DNA backbone and the amide group on the main chain of Histone proteins 3. Non-polar interactions: between the Histones and sugars on DNA 4. Salt bridges and hydrogen bonds: between side chains of basic AA (especially lys and arg) & phosphate oxygens on DNA 5. Non-specific minor groove insertions: of the H3 and H2B N-terminal tails into two minor grooves each on the DNA molecule
  10. 10. Histone tail, Histone code & Epigenetics • There are eight N-terminal domain/Tail domain in histone core. • These tail domains are heavily modified. •These modifications include:  acetylation  methylation  ubiquitylation  phosphorylation  sumoylation  ribosylation  citrullination • There are eight N-terminal domain/Tail domain in histone core. • These tail domains are heavily modified. •These modifications include:  acetylation  methylation  ubiquitylation  phosphorylation  sumoylation  ribosylation  citrullination The idea that multiple dynamic modifications regulate gene transcription in a systematic and reproducible way is called the histone code and is heritable. Mechanisms of heritability of histone state are not well understood. However it is predicted that it must be working same as DNA methylation; a histone previously modified may possess a inherent tendency to get modify as previous. This is one of the way how epigenetics works
  11. 11. The 30nm fiber • With the help of H1 the 11nm fiber compress to form more compact 30nm fiber. H1 primarily is in contact with 15-20bp of linker DNA and helps in contracting linker DNA. H1 histone is often called as ‘linker histone’ • There exist different models to explain the structure of 30nm fiber. Solenoid model and Zig-Zag model are two main models. • However recent studies demonstrates intermediate 30 nm fibers contain both the solenoid and zigzag conformations, suggesting instead that observations made in in vitro experiments might be an isolation artifact due to strictly cationic low-salt environment or chemical cross-linking (e.g., glutaraldehyde fixation). • With the help of H1 the 11nm fiber compress to form more compact 30nm fiber. H1 primarily is in contact with 15-20bp of linker DNA and helps in contracting linker DNA. H1 histone is often called as ‘linker histone’ • There exist different models to explain the structure of 30nm fiber. Solenoid model and Zig-Zag model are two main models. • However recent studies demonstrates intermediate 30 nm fibers contain both the solenoid and zigzag conformations, suggesting instead that observations made in in vitro experiments might be an isolation artifact due to strictly cationic low-salt environment or chemical cross-linking (e.g., glutaraldehyde fixation).
  12. 12. The 30nm fiber •In the one-start solenoid model, bent linker DNA sequentially connects each nucleosome cores, creating a structure where nucleosomes follow each other along the same helical path. The nucleosomes follows a chronological numbering pattern. (viz. 1,2,3…) •It is uncertain whether H1 promotes a solenoid fiber. •In the one-start solenoid model, bent linker DNA sequentially connects each nucleosome cores, creating a structure where nucleosomes follow each other along the same helical path. The nucleosomes follows a chronological numbering pattern. (viz. 1,2,3…) •It is uncertain whether H1 promotes a solenoid fiber.
  13. 13. The 30nm fiber In the two-start zigzag model, straight linker DNA connects two opposing nucleosome cores, creating the opposing rows of nucleosomes that form so called “two-start” helix. In zigzag model, alternate nucleosomes become interacting partners. (Viz. 1,3,2,4…) In the two-start zigzag model, straight linker DNA connects two opposing nucleosome cores, creating the opposing rows of nucleosomes that form so called “two-start” helix. In zigzag model, alternate nucleosomes become interacting partners. (Viz. 1,3,2,4…)
  14. 14. ‘One-start’ Helix (Solenoid)
  15. 15. ‘Two-start’ Helix (ZigZag)
  16. 16. Intermediate 30 nm fibers Four proposed structures of the 30 nm chromatin filament for DNA repeat length per nucleosomes ranging from 177 to 207 bp. Linker DNA in yellow and nucleosomal DNA in pink
  17. 17. Higher chromatin organizations (Metaphase Chromosome) • We know very less about higher chromosomal levels of genome organization • However in Histone genes it is shown that the 30nm fiber supercoils itself into six loops attached to a protein called nuclear scaffold(NS). • Even though the actual composition of the NS is not known it is shown that Topo II is a major component and is needed for the attachment of supercoiled 30nm fiber to the NS. • Several cancer chemotheraputic drugs, which are Topo II inhibitors allows strand breakage through this mechanism. • More hierarchies are also proposed. • We know very less about higher chromosomal levels of genome organization • However in Histone genes it is shown that the 30nm fiber supercoils itself into six loops attached to a protein called nuclear scaffold(NS). • Even though the actual composition of the NS is not known it is shown that Topo II is a major component and is needed for the attachment of supercoiled 30nm fiber to the NS. • Several cancer chemotheraputic drugs, which are Topo II inhibitors allows strand breakage through this mechanism. • More hierarchies are also proposed.
  18. 18. Higher chromatin organizations (Metaphase Chromosome)
  19. 19. Higher chromatin organizations (Metaphase Chromosome) Higher chromatin organizations (Metaphase Chromosome)
  20. 20. Higher chromatin organizations (Interphase Chromosome) • Determining how the Interphase chromosome is packed was a great deal to biologist. Since all the visual technologies failed to create an image of chromosome at interphase nucleus so that it explains its nature. • Two main models: • chromosome territory model, proposed by Carl Rabl in 1885. According to this model, the DNA of each chromosome occupies a defined volume of the nucleus and only overlaps with its immediate neighbors • "spaghetti" model, the DNA fiber of multiple chromosomes meanders through the nucleus in a largely random fashion, and the chromosomes are therefore intermingled and entangled with each other • Determining how the Interphase chromosome is packed was a great deal to biologist. Since all the visual technologies failed to create an image of chromosome at interphase nucleus so that it explains its nature. • Two main models: • chromosome territory model, proposed by Carl Rabl in 1885. According to this model, the DNA of each chromosome occupies a defined volume of the nucleus and only overlaps with its immediate neighbors • "spaghetti" model, the DNA fiber of multiple chromosomes meanders through the nucleus in a largely random fashion, and the chromosomes are therefore intermingled and entangled with each other
  21. 21. Higher chromatin organizations (Interphase Chromosome) • The key experiment to distinguish between two models was carried out in the early 1980s by Thomas Cremer, a German cell biologist, and his physicist brother, Christoph Cremer. • The Cremer brothers found experimental evidence that strongly supported the chromosome territory model. • The key experiment to distinguish between two models was carried out in the early 1980s by Thomas Cremer, a German cell biologist, and his physicist brother, Christoph Cremer. • The Cremer brothers found experimental evidence that strongly supported the chromosome territory model.
  22. 22. • During interphase, each chromosome occupies a spatially limited, roughly elliptical domain which is known as a chromosome territory (CT). • Each CT is comprised of higher order chromatin units of ~1 Mb each. • built up from smaller loop domains. • CT are known to be arranged radially around the nucleus. • This arrangement is both cell and tissue-type specific and is also evolutionary conserved. • The radial organization of CT was shown to correlate with their gene density and size. The gene-rich chromosomes occupy interior positions, whereas larger, gene-poor chromosomes, tend to be located around the periphery. • CT are also dynamic structures, with genes able to relocate from the periphery towards the interior once they have been “switched on”. • CT may exist either as discrete unit without intermingling or may have overlapping on each other Chromosome Territory (CT) • During interphase, each chromosome occupies a spatially limited, roughly elliptical domain which is known as a chromosome territory (CT). • Each CT is comprised of higher order chromatin units of ~1 Mb each. • built up from smaller loop domains. • CT are known to be arranged radially around the nucleus. • This arrangement is both cell and tissue-type specific and is also evolutionary conserved. • The radial organization of CT was shown to correlate with their gene density and size. The gene-rich chromosomes occupy interior positions, whereas larger, gene-poor chromosomes, tend to be located around the periphery. • CT are also dynamic structures, with genes able to relocate from the periphery towards the interior once they have been “switched on”. • CT may exist either as discrete unit without intermingling or may have overlapping on each other
  23. 23. Chromosome Territory (CT) Recurrent Clusters A) Chromosome territories (green) in liver cell nuclei (blue). B) Visualization of multiple chromosomes reveals spatial patterns of organization. Chromosomes 12 (red), 14 (blue), and 15 (green) form a triplet cluster in mouse lymphocytes. Part A: © 2004 Parada, L. A. et al. Tissue-specific spatial organization of genomes. Genome Biology 5:R44 doi:10.1186/gb-2004-5-7-r44. Part B: © 2002 Cell Press/Elsevier Inc. Parada, L. A. et al. Conservation of relative chromosome positioning in normal and cancer cells. Current Biology 12, 1692–1697 (2002).
  24. 24. Chromosome Territory (CT) • Large areas of chromosomal identity between different species that have been maintained throughout evolution. These areas of identity maintain their positions in different species (Tanabe et al., 2002). • CT can reposition in disease, which might provide novel insights into disease mechanisms and why genes are incorrectly expressed in disease. • Scientists have manipulated the localization of chromosomes and seen some changes in gene expression as a result, thus suggesting a possible mechanism for the connection between CT and disease (Finlan et al., 2008). • No proteins have been identified that either anchor chromosomes in the nucleus or link multiple chromosomes to each other to establish chromosome clusters. • Large areas of chromosomal identity between different species that have been maintained throughout evolution. These areas of identity maintain their positions in different species (Tanabe et al., 2002). • CT can reposition in disease, which might provide novel insights into disease mechanisms and why genes are incorrectly expressed in disease. • Scientists have manipulated the localization of chromosomes and seen some changes in gene expression as a result, thus suggesting a possible mechanism for the connection between CT and disease (Finlan et al., 2008). • No proteins have been identified that either anchor chromosomes in the nucleus or link multiple chromosomes to each other to establish chromosome clusters.
  25. 25. Chromosome Territory (CT) Movement of CT GENE OFF GENE ON
  26. 26. Chromosome Territory (CT) FISH of Human interphase nucleus 10µm
  27. 27. Other domains in nucleus • Transcription factories – transcription is spatially organized into discernable nuclear structures in which multiple RNA polymerases and active genes dynamically localize into nuclear bodies termed transcription factories. • Transcription factories – transcription is spatially organized into discernable nuclear structures in which multiple RNA polymerases and active genes dynamically localize into nuclear bodies termed transcription factories.
  28. 28. Molecular Models of looping • Random loop Model oWith loops at all scales > 150bp • Multi-loop model oExplains 120kbp rosette Structure • Random Walk/ Giant loop Model oThe basic feature of the RW-GL model is the existence of 1-3 Mbp size loops along a randomly oriented backbone • Random loop Model oWith loops at all scales > 150bp • Multi-loop model oExplains 120kbp rosette Structure • Random Walk/ Giant loop Model oThe basic feature of the RW-GL model is the existence of 1-3 Mbp size loops along a randomly oriented backbone Looping allows spatial closeness of regulatory elements thus explaining how it functions at 10s of Kbps and is demonstrated in β-globin genes
  29. 29. Sequential organization
  30. 30. Sequential organization
  31. 31. Tandem repeats Microsatellite DNA • Unit - 2-4 bp (most 2). • Repeat - on the order of 10- 100 times. • Location - Generally euchromatic. • Examples - Most useful marker for population level studies.. Minisatellite DNA • Unit - 15-400 bp (average about 20). • Repeat - Generally 20-50 times (1000-5000 bp long). • Location - Generally euchromatic. • Examples - DNA fingerprints. Tandemly repeated but often in dispersed clusters. Also called VNTR’s (variable number tandem repeats). • Tandem repeats occur in DNA when a pattern of two or more nucleotides is repeated and the repetitions are adjacent to each other • Form different density band on density gradient centrifugation (from bulk DNA) -satellite • Unit - 2-4 bp (most 2). • Repeat - on the order of 10- 100 times. • Location - Generally euchromatic. • Examples - Most useful marker for population level studies.. • Unit - 15-400 bp (average about 20). • Repeat - Generally 20-50 times (1000-5000 bp long). • Location - Generally euchromatic. • Examples - DNA fingerprints. Tandemly repeated but often in dispersed clusters. Also called VNTR’s (variable number tandem repeats).
  32. 32. Interspersed Repetitive DNA • Interspersed repetitive DNA accounts for 25–40 % of mammalian DNA. • They are scattered randomly throughout the genome. • The units are 100 – 1000 base pairs long. • Copies are similar but not identical to each other. • Interspersed repetitive genes are not stably integrated in the genome; they move from place to place. • They can sometimes mess up good genes These are: • Retrotransposons (class I transposable elements) (copy and paste), copy themselves to RNA and then back to DNA (using reverse transcriptase) to integrate into the genome. • Transposons (Class II TEs) (cut and paste) uses transposases to make makes a staggered sticky cut.
  33. 33. Interspersed Repetitive DNA • Retrotransposons are:  long terminal repeat (LTR) Any transposon flanked by Long Terminal Repeats. (also called retrovirus-like elements). None are active in humans, some are mobile in mice.  long interspersed nuclear elements (LINEs) encodes RT and  short interspersed nuclear elements (SINEs) uses RT from LINEs. example Alu made up of 350 base pairs long, recognized by the RE AluI (Non-autonomous) • Retrotransposons are:  long terminal repeat (LTR) Any transposon flanked by Long Terminal Repeats. (also called retrovirus-like elements). None are active in humans, some are mobile in mice.  long interspersed nuclear elements (LINEs) encodes RT and  short interspersed nuclear elements (SINEs) uses RT from LINEs. example Alu made up of 350 base pairs long, recognized by the RE AluI (Non-autonomous)
  34. 34. Gene rich regions have been visualized with a fluorescent probe that hybridizes to the Alu interspersed repeat, which is present in more than a million copies in human genome. For unknown reasons, these sequences cluster in chromosomal regions rich in genes(GREEN). In this picture regions depleted for these sequence are RED, while the average regions are YELLOW. The gene rich regions are seen to be depleted in the DNA near the nuclear envelope. A. Bolzer et. al, PLoS Biol. 3:826- 842, 2005 Linking sequential organization and Genome Organization Gene rich regions have been visualized with a fluorescent probe that hybridizes to the Alu interspersed repeat, which is present in more than a million copies in human genome. For unknown reasons, these sequences cluster in chromosomal regions rich in genes(GREEN). In this picture regions depleted for these sequence are RED, while the average regions are YELLOW. The gene rich regions are seen to be depleted in the DNA near the nuclear envelope. A. Bolzer et. al, PLoS Biol. 3:826- 842, 2005 5µm
  35. 35. Thank youThank you

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