Chapter 10 - Chromatin Flashcards

(68 cards)

1
Q

Mass Composition of Chromatin

A

DNA: 30%
Protein: Histones: 30%
Protein: Non-Histone: 30%
RNA: 10%

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2
Q

Hierarchy of DNA Organization ensures

A

That the DNA is packaged in the most compact state.

dsDNA -> Wrapped 6x into nucleosomes -> 10 nm fiber

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3
Q

Nucleosomes

A

Eight small proteins that organize ~200 bp of DNA into a bead-like structure

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4
Q

Histones

A

The proteins that constitute the inner core of the nucleosome around which the DNA is wrapped

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5
Q

• Packaging of the DNA into a nucleosome generates

a chromatin fiber that is ______ in diameter.

A

About 10 nm

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6
Q

Hierarchy of DNA Organization ensures

A
That the DNA is packaged in the most compact state.
dsDNA -> 
Wrapped 6x into nucleosomes -> 
Wrapped 40x into 10 nm fiber -> 
Wrapped 1,000-10,000x into 30 nm fiber
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7
Q

• Packaging of the DNA into a nucleosome generates

a chromatin fiber that is ______ in diameter.

A

About 10 nm

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8
Q

30 nm fiber is

A

Coiled into a helical structure that includes about 6 nucleosomes per turn. 30 nm in diameter.

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9
Q

30 nm fiber is

A

Coiled into a helical structure that includes about 6 nucleosomes per turn. 30 nm in diameter.

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10
Q

DNA can be:

A

Nucleosome-bound or Linker DNA

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11
Q

MNase

A

The exposed linker DNA is the cleavage site of the micrococcal nuclease (MNase)

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12
Q

MNase

A

The exposed linker DNA is the cleavage site of the micrococcal nuclease (MNase).
Demonstrate that >95% of DNA is nucleosome bound.

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13
Q

Typically _______ bp DNA wraps around a nucleosome.

A
  • Typically ~200 bp DNA wraps around a nucleosome.

* Varies from 154-260 bp between species and/or mammal tissue.

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14
Q

Nucleosome

A

Histones form a cylindrical structure:
Diameter of 11 nm
Height of 6 nm
DNA wraps 1.66 turns around the outside of the nucleosome.
DNA enters and exits within close proximity.
Sites separated by ~80 bp are also in closer proximity due
to “wrapping” of the DNA.

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15
Q

Core DNA

A

Directly interacts with the histone octamer, is resistant

to MNase digestion, and consistently 146 bp.

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16
Q

Linker DNA

A

Located between histone octamers, digested by MNase, and includes 8-114 bp.

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17
Q

Nucleosome

A

Histones form a cylindrical structure:
Diameter of 11 nm
Height of 6 nm
DNA wraps 1.66 turns around the outside of the nucleosome.
DNA enters and exits within close proximity.
Sites separated by ~80 bp are also in closer proximity due
to “wrapping” of the DNA.

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18
Q

Linker DNA

A

Located between histone octamers, digested by MNase, and includes 8-114 bp.

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19
Q

Each histone includes

A

3-α helices and 2-interconnecting loops (called the histone

fold)

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20
Q

Histone complex formation

A
  • H3 and H4 form a tetramer (H32-H42)

- H2A and H2B form a dimer (H2A-H2B)

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21
Q

H32-H42 tetramer form

A

“Horseshoe” and constitutes the 11 nm diameter of the nucleosome.

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22
Q

H2A-H2B dimer

A

Bind each end of the H32-H42 tetramer “horseshoe.”

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23
Q

H2A-H2B dimer

A

Bind each end of the H32-H42 tetramer “horseshoe.”

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24
Q

Linker histones:

A
  • H1
  • Functions to “seal” DNA in the nucleosome with its location at the site where DNA “enter/exits”
  • Functions to promote 30 nm fiber
  • Can be extracted from chromatin without affecting nucleosome structure.
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25
Core Histones include:
1. Globular core 2. N- and/or C- terminal tail - N-terminal tail: H3, H4, H2A, and H2B - C-terminal tail: H2A and H2B
26
The amino acid on the histone tail is subject
To covalent modification or protein post-translational modification (PTM). The lysine amino acid is a common site for multiple modifications.
27
The amino acid on the histone tail is subject
To covalent modification or protein post-translational modification (PTM). The lysine amino acid is a common site for multiple modifications.
28
PTM includes
Adding a functional chemical group: P, Ac, Me. Affect association with DNA and chromatin structure. Commonly a given histone is modified at multiple sites simultaneously.
29
HAT
Histone acetyltranferase
30
HDAC
Histone deacetylase
31
H3K4 Methylation
Transcription activation
32
H3K9 Methylation
Chromatin condensation. Don't transcribe. Required for DNA methylation.
33
H3K9 Acetylation
Transcription activation
34
H3S10 Phosphorylation
Transcription activation
35
K3K14 Acetylation
Prevents methylation at K9. | Thereby prevents chromatin condensation.
36
H3K79 Methylation
Telomeric silencing
37
H4K5 Acetylation
Nucleosome assembly
38
H4K12 Acetylation
Nucleosome assembly
39
H4K16 Acetylation
Nucleosome assembly
40
H4K16 Acetylation
Fly X activation
41
How does histone modification initiate changes in | chromatin structure?
1. Changes the charge of the histone. | 2. Provides a binding site for non-histone chromatin remodeling proteins.
42
How does histone modification initiate changes in | chromatin structure?
1. Changes the charge of the histone. | 2. Provides a binding site for non-histone chromatin remodeling proteins.
43
H2A
Canonical core histone
44
H2AZ
Function in gene expression, chromosome segregation
45
H2AX
Function in DNA repair and recombination | Major core histone in Yeast
46
MacroH2A
Function in X-chromosome inactivation, transcription repression
47
H2B
Canonical core histone
48
H3
Canonical core histone
49
CenH3
Function in kinetochore assembly
50
CenH3/CENP-A
Function in kinetochore assembly | Mark of a functional centromere
51
Gamma-H2AX
The phosphorylated form of the histone variant H2AX. DNA Damage Machinery initiates phosphorylation of the H2AX variants. Antibody for gamma-H2AX identifies damaged DNA.
52
Two features can influence the position of a nucleosomes in the DNA:
1. Ability to bend the DNA region around the nucleosome (DNA flexibility) - the minor groove in contact with the nucleosome is substantially compressed (A-T rich) - minor groove outside surface less compressed (G-C rich) 2. Presence of other specific proteins with the DNA
53
DNase I and DNase II Treatment
Cut a single strand of the DNA duplex at the minor groove to generate repeat lengths which reflect the helical turn. Shows rotational positioning
54
MNase Treatment
MNase cuts double strands of DNA between nucleosomes. Show translational positioning Generates an MNase Map of the nucleosome positions in a gene. A select probe can be generated to identify the TATA sequence.
55
Nucleosomal DNA _____ bp per turn
10.1 bp/turn Nucleosomal DNA is less tightly wound than DNA in solution
56
DNA in solution ______ bp per turn
10.5 bp/turn
57
DNase Hypersensitive Sites
Found at the promoters of expressed genes. Generated by the binding of transcription factors that exclude histone octamers.
58
DNase I and DNase II Treatment
Cut a single strand of the DNA duplex at the minor groove to generate repeat lengths which reflect the helical turn. Shows rotational positioning
59
Nucleosomal DNA _____ bp per turn
10.1 bp/turn
60
DNase Hypersensitive Sites
Found at the promoters of expressed genes.
61
Nucleosome position at a locus control region (LCR) can dictate:
Transcription efficiency of a gene cluster. LCR facilitates chromatin remodeling to ensure that the transcription machinery can bind the individual genes.
62
Replication-dependent pathway
• Histone Chaperone proteins include: CAF1 and/or ASF1 • Replication Machinery recruits CAF1/ASF1 • H32-H42 tetramers bind the new daughter strand with assistance of chaperone. • H2A-H2B dimers bind the incorporated tetramers
63
Replication-dependent pathway:
* Histone Chaperone proteins include: CAF1 and/or ASF1 * Replication Machinery recruits CAF1/ASF1 • H32-H42 tetramers bind the new daughter strand with assistance of chaperone. • H2A-H2B dimers bind the incorporated tetramers
64
Nucleosome Assembly grouped into two distinct pathways:
1. Replication-dependent pathway | 2. Replication-independent pathway (occurs during transcription or DNA repair)
65
Two insulators limit
Transcriptional activation or repression in the gene loci between the insulators. Ex. Polytene puffs.
66
Replication-independent pathway:
• Different Histone Chaperone proteins include: HIRA • Different Transcription or DNA Repair Machinery recruits Histone Chaperone • Different H3.32-H42 tetramers bind the new daughter strand with assistance of chaperone. • H2A-H2B dimers bind the incorporated H32-H42 tetramers
67
Replication-independent pathway:
• Different Histone Chaperone proteins include: HIRA • Different Transcription or DNA Repair Machinery recruits Histone Chaperone • Different H3.32-H42 tetramers bind the new daughter strand with assistance of chaperone. • H2A-H2B dimers bind the incorporated H32-H42 tetramers
68
Linker Number Paradox
The discrepancy can be accounted for by the difference | in the number of bp/turn of nucleosomal (10.5 bp/turn) versus free DNA (10.1 bp/turn).