Chromosome

Chromatin
Telomeres

周金秋
jqzhou@sibs.ac.cn
Istitute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
Fall, 2005
I. Chromosome and Chromatin

II. Centromere

III. Telomeres
I. Chromosome and Chromatin

II. Centromere

III. Telomeres
Genome size in different organisms
 Genomes and gene number

6000 19,000 13,500 20,000

32 12 8 25

30,000 25,000
36 40 30,000 10
46
 Compacting DNA
into chromosomes is essential
3 x 109 bp
3.4 Å

~1.0 meters / haploid genome

~2.0 meters / per cell

~10 micrometers - nucleus diameter

~ 1 micrometer – condensed chromosomes

>10,000 fold
 History of chromatin and chromosomes
1673 Van Leeuwenhoek (Dutch) Microorganisms
1665 Hooke (England) Cell
Early 1800s Brown (Scottland) Nucleus (little nut)
1866 Mendel (Austria) Mendel’s Law
1869 Miescher (Swiss) Nuclein (C, H, O, N, P)
1857 Perkin (England) aniline purple (mauveine dye)
1879 Flemming (Germany) Chromatin, chromosome, Mitosis (thread 1882)
1887 Van Beneden (Belgium) Chromosome No
1900 De Vries (Neitherland) Re-discovery of Mendel law
Correns (Germany)
Tschermak (Austria)
1902-1903 Sutton (USA) Chromosomes are paired and may be the
Boveri (Germany) carriers of heredity. Mendel's "factors" (genes)
are located on chromosomes
1920s-1930s Morgan (USA) Morgan Law
1938 Mullar (USA) Mutation of chromosome
1928 Griffith (Britain) Transforming principle
1943 Avery (Canada) Genetic material - DNA
 A-T rich G band

Centromere

Large rDNA
Chromosome karyotype (human)
Chromosome spread and FISH
Visualize
chromosomes
(FISH)

Fluorescent antibody-tagged DNA probes hybridize to

their complementary sequences in the chromosomes.
Human cells contain 23 pairs of Chromosomes. For each pair
of chromosomes, one is maternal and one is paternal –
homologous chromosomes Sex chromosomes are non-
homologous chromosomes, X from mom, Y from dad.
Visualize gene(s)
(FISH)

Chromosome
spread
(FISH)
Visualize gene(s)
(FISH)
Visualize gene(s)
(FISH)

Chromosome
Translocation
(FISH)
Function of chromosome and chromatin

• Storage of genetic information

• Precise segregation of replicated DNA into
two daughter cells
• Platform for transcription, replication,
recombination and repair

Problem(s)
How to retrieve genetic information from
DNA packaged in chromosomes?
I. Chromosome and Chromatin

II. Centromere

III. Telomeres
 Chromatin in different cell-stage

Interphase chromatin A mitotic chromosome

Euchromatin: (1) delicate
(2) active
(3) at the nucleus interior
,
Heterochromatin: (1) darkly staining
(2) tightly packaged,
(3) genetically inactive.
(4) at the nucleus periphery
Constitutive heterochromatin:
fixed and irreversible
Centromere, Telomeres (Movie 1)

Facultative heterochromatin:
able to return to the euchromatin
inactive X chromosome
Chromatin composition:

DNA Stable association

Histone proteins

Nonhistone:
HMG proteins
residual proteins
phosphoproteins

RNA species
lipid species
 Nucleosome

Nucleosome: a nucleosome core particle

+ linker DNA (180-200 bp)
+ a linker histone

Nucleosome core particle:

histone octamer (2x H2A, H2B, H3, H4)
+ 146 bp DNA
 Nucleosome: unit of chromatin

A) 30 nm fibers
B) beads on a string-nucleosome
From interphase nucleus
Nucleosome: unit of chromatin
Histone depleted metaphase
chromosomes
Nucleosomes can be
isolated by digesting
with nucleases that cut
between the nucleosomes
in a region called the linker
Nucleosome-octamer
2 each H2A, H2B, H3, H4
Histones - highly basic proteins

Protein Molecular Major

weight Amino acid
++
H1 21 Lys
H2a 13.8 Lys
H2b 13.8 Lys
H3 15.4 Arg/Lys
H4 11.4 Arg/Lys
The position of the core histone in the
nucleosomes
Nucleosome core particle
Crystal structure of the mono-nucleosome

From Luger et al Nature 389: 251 - 260 (1997)

142 hydrogen bonds between DNA and nucleosome,

mostly between phosphodiester bonds and amino acid
backbone of histones
Is DNA in the nucleosome different from DNA in solution?
1. DNA (146 bp) is wrapped in 1.75 left-handed superhelical turns
2. One side of DNA is in contact with histone octamer
3. DNA helical turns in a nucleosome have an average number of
base pairs per helical turn of 10.2 vs 10.5 of DNA in solution
Histone tail interactions with DNA

From Luger et al Nature 389: 251 - 260 (1997)

Histone fold-
3 alpha helices
and 2 folds
N terminal tails are
subject to covalent
modification-important
for transcription
Histone self-assembly
Modification of histone tail
Silent chromatin
 Gene silencing and silent chromatin

Gene silencing: gene silencing acts in a regional rather than

promoter- or sequence-specific manner to generate large domains
or DNA that are usually inaccessible to DNA binding proteins: RNA
polymerase, cellular recombination machinary, exogenous
enzymes (dam methyltransferase and restriction endonuclease).

Silent chromatin domain is persistent through mitotic and

meiotic cell divisions such that a particular chromatin structure
(DNA and its associated proteins) is replicated during the process
of chromosome duplication. This mode of inheritance, commonly
referred to as epigenetic inheritance, is believed to underlie
cellular memory mechanisms that maintain cell identity and stable
patterns of gene expression in eukaryotes.
 Silent chromatin and heterochromatin

Silent chromatin shares the central properties of general

inaccessibility and epigenetic in heritance with
heterochromatin. Therefore, although silent chromatin
domains, unlike heterochromatin, are not always
cytologically distinguished, they are often referred to as
heterochromatic.

Gene silencing and heterochromatin are often

associated with repetitive DNA sequences and may be
involved in stabilizing such sequences.
 Biochemical nature of
silent chromatin/heterochromatin

1. Histone H3 methylated
at lysine 9 (H3-mLys9)

2. Hypoacetylation of
lysine residues

3. cytosine methylation,
the most common
form of DNA
modification in
eukaryotes.

Richards and Elgin (2002) Cell 108:489

Telomere heterochromatin/Position effect

Telomere looping

Rap1p Nucleosome Sir3p Sir4p

Strahl-Bolsinger et al Genes & Development 1997
Telomere silencing of ADE2
Telomere position effect
ADE2 “OFF” = RED
ADE2 ADE2 “ON” = WHITE

Wildtype, ADE2 gene near

telomere is silenced

Lack of a telomere binding

protein reduces/disrupts
telomere silencing

Ivessa et at, (2002) G&D16:1383

Assembly of silent chromatin in budding
yeast
(example of biochemical study)
protective
nucleosomes telosome cap

report gene

Rap 1 Sir2/3/4 complex Ku Cdc13

Chromatin immunoprecipitation
(ChIP)
DNA-binding proteins are
crosslinked to DNA with
formaldehyde in vivo.

Isolate the chromatin. Shear

DNA along with bound
proteins into small fragments.

Bind antibodies specific to the

DNA-binding protein to isolate the
complex by precipitation. Reverse
the cross-linking to release the
DNA and digest the proteins.

Use PCR to amplify specific

DNA sequences to see if they
were precipitated with the
antibody.
 Sir2/3/4 interacts with telomeric DNA
Sir2/Sir3 and DNA interaction requires Sir4

Luo et al (2002) Genes & Development 16:1528

Sir2/Sir3/Sir4 binding to telomeric DNA
decreases at telomere distal regions

Luo et al (2002) Genes & Development 16:1528

Sir2 and Sir3 binding at the telomeric end require
the enzymatic activity of Sir2
Model for assembly of silent chromatin
in budding yeast

Moazed (2001) Mol Cell 8:489

Model for assembly of silent chromatin
domain in fission yeast

Moazed (2001) Mol Cell 8:489

 The Swi6/HP1 silencing complex is
conserved in the fission yeast, S. pombe,
and metazoans

Moazed (2001) Mol Cell 8:489

Silencing components in different systems

Richards and Elgin (2002) Cell 108:489

Euchromatin and heterochromatin

Richards and Elgin (2002) Cell 108:489

Chromosome Packing
 How linear DNA molecule
is packaged into a compact chromosome?

Interphase chromatin A mitotic chromosome

?
Interphase M phase
“Beads on a string” to 30 nm Chromatin Fiber?
Histone H1 - linker of nucleosomes
 Zigzag model
10 nm nucleosome ------ 30 nm fiber
 Solenoid Model
six to eight nucleosomes per turn
 Linker histones in higher
order chromatin compaction
 Further Compaction?
Chromatin in the interphase nucleus is believed to organized
into discrete domains defined by sites of attachment to the
nuclear matrix.
Scaffold attachment regions (SARs)

• Regions of the chromosomes with sequences

specific for topoisomerase, HMG protein, and
histone H1 binding
• Found only in untranscribed regions of the
eukaryotic chromosomes
• Spaced along the chromosomes, with the
intervening regions containing one or more
genes?
• Highly AT rich (65%) and may be several
hundred bp long
 How linear DNA molecule
is packaged into a compact chromosome?

???
I. Chromosome and Chromatin

II. Centromere

III. Telomeres
 Centromere

Centromere
Figure 23-38, p. 1094, Molecular Cell Biology, 3rd ed., Lodish, et al.
Centromere is a region of a eukaryotic
chromosome where the kinetochore is
assembled. It is the site where spindle fibers of
the mitotic spindle attach to the chromosome
during mitosis. It is the site at which a chromatid
and its identical sister attach together during the
process of cell division. It is a chromosomal
locus that ensures delivery of one copy of each
chromosome to each daughter at cell division.

In most eukaryotes, the centromere has no

defined DNA sequence. It typically consists of
large arrays of repetitive DNA where the
sequence within individual repeat elements is
similar but not identical.
Kinetochore, the protein complex
assembled at each centromere,
serves as the attachment site for
spindle microtubules and the site at
which motors generate forces to
power chromosome movement.
 Functions of centromere

• Required for chromosome stability

• Sister chromatid pairing

• Mitotic and meiotic spindle attachment

• Chromosome movement

• Cell cycle checkpoint control

 The structure of centromere
• 1 centromere / chromosome
• Structural complex
• Kinetochore - spindle fiber attachment
• DNA at yeast centromeres is relatively simple
• Human centromere is a family of highly
repeated, tandemly arrayed ‘satellite’ DNA
which measure 300-5,000 kb in length Repeat
sequences
• Specific associated proteins
Centromere DNA

Fukagawa.(2004) Chromosome Res. 12: 557–567

Centromere DNA

Bjerling and Ekwall. (2002) Braz J Med Biol Res. 35: 499-507
Centromere proteins

Bjerling and Ekwall. (2002) Braz J Med Biol Res. 35: 499-507
 Budding yeast kinetochore proteins
and their homologues

Essential genes in red

nonessential genes in black
metazoan homologue in black (right column)
S. pombe homologue in blue.
Cheeseman et al. (2002) J Cell Biol. 157: 199-203
 S. pombe Centromere

- Swi6 and Cnp1 localization

SpCENP-A

,Kniola et al, (2001) MBC 12:2767–2775

 S. pombe Centromere
- Ndc80 and Cnp1 localization

Kniola et al, (2001) MBC 12:2767–2775

Clr4p and Rik1p are required for centromere
localization of Swi6p

Ekwall et al, (1996) J. Cell.

Sci. 109,2637–2648.
Centromere dysfunction in clr4 and rik1 mutants

WT

clr4

rik1
Ekwall et al, (1996) J. Cell.
Sci. 109,2637–2648.
Centromere-associated proteins in yeast

Mellone and Allshire. (2003) Curr Opin Genet Dev. 13:191–198

Centromere DNA of higher eukaryotes

• The universal presence of a great

abundance of tandemly repeated DNA

• The size of centromere DNA varies

from several hundreds of kilobases to
tens of megabases on each
chromosome

• Lack of sequence conservation

CENP-A proteins form a higher order
structure in Drosophila and humans
Drosophila
CID (green, CENP A homologue)
ROD (red, outer kinetochore protein,
CENP E homologue)

Human
CENP A (green, histone H3-like protein)
CENP E (red, outer kinetochore protein)

Human
CENP A (green, histone H3-like protein)
CENP C (red, inner kinetochore protein)

Blower et al, (2002) Developmental Cell 2:319–330

CID, CENP-A H2A, H2B, H3 in Extended
Chromatin Fibers

H2AB (green)
CID (red)

H2AB (green, continuous)

CID (red, discontinuous)

CID (red)
Histone H3 (green)

CID (red)
Histone PH3 (green)

Human CENP-A (green)

H3 (red), interspersed
Blower et al, (2002) Developmental Cell 2:319–330
Models for 3D organization of centromeric
chromatin in Drosophila and humans

Solenoid model

Looping model

Blower et al, (2002) Developmental Cell 2:319–330

Organization of a human or mouse centromere

Choo. (2000) Trends Cell Biol. 10: 182-188

Centromeric organization
of fission yeast and human.

Yanagida (2005) Phil. Trans. R. Soc. B 360, 609–621

I. Chromosome and Chromatin

II. Centromere

III. Telomeres
See You Next Time