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							The Nucleus: Chromosome Structure & Chromatin
The Nucleus: - has inner & outer membranes: nuclear envelope - perforated by nuclear pores: elaborate gates that control transport of small and large molecules to/from nucleus - connected to endoplasmic reticulum Nuclear Lamina: - composed of proteins called ―nuclear lamins’ (A, B, C) - form intermediate filaments - possible attachment point for chromatin & nuclear RNAs
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Nucleus: the components

- endoplasmic reticulum (ER): shares nuclear membrane (perinuclear space); site where ribosomes ‘inject’ newly-made proteins that are destined to be incorporated into membranes (e.g., cell surface) or be secreted - nuclear pore complex (NPC): highly complex proteinaceous pore that regulates the entry/exit of proteins, and exit of mRNA -nuclear lamina: provides structural rigidity to nucleus and possible sites of attachment for chromatin, etc. -nuclear matrix: diffuse nuclear scaffold consisting of proteins (e.g. actin) - nucleoplasm: chromatin/ chromosome-containing region nucleolus: a ribosome-producing ‘sub-compartment of the nucleus

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The Nucleolus: - specialized region of the nucleus with densely stained chromatin in electron micrographs - site of rRNA genes & of ribosome assembly - rRNA transcripts  about 80 proteins (from the cytoplasm)  many RNAs -small and large ribosome subunits (40S, 60S) are assembled and exported to the cytoplasm separately Chromatin: is the physical state of chromosomes during interphase; it is a complex consisting of histones, nonhistone chromosomal proteins and DNA. - But, during metaphase, chromatin (which is a relatively loose & spread-out structure in interphase nuclei) becomes extensively folded or ‘condenses’ to form the familiar metaphase chromosomes
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Overview of Chromatin Structure

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Why Chromatin?
Each eukaryotic chromosome contains a continuous DNA chain along its entire length –can be up to ~10 cm long for human chromosomes (~ 3.5 x 108 bp). Therefore the DNA must be organized in very elaborate ways in order to package or condense it into a manageable size, so that it will fit into the nucleus- accomplished by different levels of chromatin packaging.
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Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11(or 10) nm
chromatin fiber-fundamental unit of chromatin (“beads on a string”); nucleosomes consist of histones.

30 nm chromatin fiber or solenoid: created by
coiled 11 nm fiber- interphase chromatin exists as highly condensed solenoid with interspersed extended loops containing transcribed genes. Metaphase chromosomes: further looping/coiling of solenoid DNA into highly compacted, transcriptionally silent, form of chromatin- occurs during mitosis.
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Levels of Chromatin Organization
DNA nucleosome (11 nm Fiber) 30 nm fiber
physiologically important structure

Loops compacted metaphase chromosome

?

Loops
These are only models at this stage

The loops of 30 nm fibers contain 20,000 – 100,000 bp (i.e., about the sizes of genes) Note that other DNA and chromatin-binding proteins (besides histones) are also involved in organizing chromatin.
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Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11 (or 10) nm
chromatin fiber-fundamental unit of chromatin (“beads on a string”); nucleosomes consist of histones.

30 nm chromatin fibre or solenoid: created by
coiled 11 nm fiber- interphase chromatin exists as highly condensed solenoid with interspersed extended loops containing transcribed genes. Metaphase chromosomes: further looping/coiling of solenoid DNA into highly compacted, transcriptionally silent, form of chromatin- occurs during mitosis.
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Histones (Components of the nucleosome)
The major protein component of chromatin. - small, very basic (lys, arg) proteins (to neutralize the highly charged DNA) - assembly of histones with DNA requires, a molecular chaperone, nucleoplasmin

Five Types of Histones:
“Core” Histones

H1 H2A H2B H3 H4

Clamp for DNA wrapped around nucleosome Found in 2 copies of each in the nucleosome; The DNA is then wrapped around the protein core made of these proteins -- together they form an octamer

H4:

H3:
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Is very highly conserved between species (98% identical between cows and peas) ~ 1% change in 600 million years Is also very conserved (97% identical)
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H1
Linker histone

HISTONES
are highly conserved, small, basic proteins
helix

H2A

H2B
Core histones
variable

H3 H4
N

conserved

Histone acetylation is a reversible modification of lysines in the N-termini of the core histones. Result: • reduced binding to DNA • destabilization of chromatin

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Experimental evidence for nucleosomal packaging of DNA into 11 nm chromatin fibre

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Nucleosomal packaging of DNA

In the presence of Histone H1, 175-200 bp DNA is associated with the nucleosome- but, only 146 bp is wrapped around the octamer (i.e. if H1 is removed, ~146 bp is observed in nuclease digests) Histone H1- helps ‘clamp’ the DNA onto the nucleosome and participates in higher-order chromatin folding
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dsDNA wraps around the nucleosome two times.

Histones

dsDNA

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Compaction of DNA in Nucleosomes
Each nucleosome serves to compact DNA Compaction ratio: 68 nm / 10 nm = ~ 7 (DNA 200 bp / nucleosome)

Length of fully extended 200 bp of DNA ~ 68 nm
However, total condensation of metaphase chromosomes:

1 m  100 m (104 packing ratio)
- therefore nucleosomes only play a small part

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Histone modifications
Histones are also modified in order to regulate:

1. chromosome structure; compaction
2. gene activity The main types of modifications are: a) Phosphorylation of serines b) Methylation of lysines c) Acetylation of lysines: neutralizes + charges

--NH3+  --NH—C—CH3
O
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Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11 (or 10) nm
chromatin fiber-fundamental unit of chromatin (“beads on a string”); nuclesomes consist of histones.

30 nm chromatin fibre or solenoid: created by
coiled 11 nm fiber- interphase chromatin exists as highly condensed solenoid with interspersed extended loops containing transcribed genes. Metaphase chromosomes: further looping/coiling of solenoid DNA into highly compacted, transcriptionally silent, form of chromatin- occurs during mitosis.
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11 nm fiber is coiled into 30 nm Solenoid- requires histone H1 DNA

Schematic of formation of 30nm chromatin fiber

Histones

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Chromatin fibers

30 nm chromatin fiber

11 nm (beads)

+ charged N termini (bind DNA on neighboring nucleosomes) Solenoid: • HIGH level of histone H1 “pull” nucleosomes into solenoid

highly acetylated core histones (especially H3 and H4) Short extended regions can exist between solenoids: • Reduced level of histone H1 • Gene transcription possible
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• NO gene transcription in solenoid
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Interphase chromosomes: mosaics of
solenoids/compacted solenoids and extended chromatin loops I. Amphibian Lampbrush Chromosomes: consist of elaborately folded 30 nm fibers and extended loops containing expressed genes

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II. Insect Polytene Chromosomes: consist of >1000 precisely aligned chromatidscontain condensed and decondensed regions
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Heterochromatin versus Euchromatin Interphase chromosomes consist of two types of chromatin (their differential assembly is due largely to the association of specific kinds types of non-histone proteins with nucleosomes and DNA): Euchromatin: less condensed chromatin domains- comprises most of chromosome “arms”; only clearly visible during mitosis; contains high density of transcribed genes (only ~10% are active at a given time). Heterochromatin: highly condensed chromatin domainslocated near the centromere and telomeres; remains compacted/condensed throughout cell cycle; contains low density of transcribed genes and can cause “silencing” of relocated euchromatic genes.
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Heterochromatic regions of insect polytene chromosomes coalesce into “chromocentre”

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Different Levels of Chromatin Organization
Nucleosomes package DNA into the 11(or 10) nm
chromatin fiber-fundamental unit of chromatin (“beads on a string”); nuclesomes consist of histones.

30 nm chromatin fibre or solenoid: created by
coiled 11 nm fiber- interphase chromatin exists as highly condensed solenoid with interspersed extended loops containing transcribed genes.

Metaphase chromosomes: further looping/coiling
of solenoid DNA into highly compacted, transcriptionally silent, form of chromatin-occurs during mitosis.
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Typical Metaphase Chromosome
-this structure allows the easy separation of sister chromatids and protects the fragile DNA molecules during mitosis
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Consists of loops/coils of chromatin attached to a protein network or scaffold
Primary Constriction: contains centromere

FIG. 1
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The Metaphase Chromosome
-formed by looping and coiling of condensed chromatin: assoc. with H1 phosphorylation

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Topoisomerase II is associated with loops (30 nm) – suggests individual loops may be autonomously supercoiled  Allows for independent regulation of supercoiling & chromatin structure for specific regions

Even higher-order organization is required than the higher-order loops for metaphase condensation  Protein scaffolds appear to be involved  Regulated (?) by Histone H1 phosphorylation - 5 serine residues phosphorylated during condensation
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Summary
2 nm – DNA double helix

11 nm – Nucleosome (11 nm fiber)
30 nm – 30 nm Fiber Interphase chromatin: genes expressed in extended loops (11nm fibers)

300 nm – Loops I

700 nm – Loops II 1400 nm – chromosome
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Condensation into metaphase chromosomes
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Figure 8.30 – Alberts, Mol. Biol. Cell.

Summary of Principal Types of Histone Modifications and Their Roles in Chromatin Packaging: occur in N-terminal “tails”
1. Acetylation of lysines: carried out by histone acetyl transferases (HATs); acetyl groups removed by histone deacetylases (HDACs)acetylation neutralizes lysine + charges and “loosens” histone/DNA interactions - promotes chromatin decondensation

2. Methylation of lysines: carried out by histone methyl transferasespromotes formation of highly compacted chromatin e.g. heterochromatin 3. Phosphorylation of serines: carried out by kinases; phosphatases remove phosphates; 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.

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