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Chromosome, Chromatin and Telomere

Jiemin Wong, Ph.D.


Associate Professor
Department of Molecular and Cellular Biology
Baylor College of Medicine
Houston TX 77030
Jwong@bcm.tmc.edu
713-798-6291 (phone)
713-790-1275 (fax)
I. Chromosome and Chromatin

II. Modulation of Chromatin Structure

III. Telomere and its Implication in Aging and

Cancer
I. Chromosome and Chromatin

Eukaryotic DNA is packaged into


a set of chromosomes
Why compaction of DNA into chromosomes is essential?
Genomes and Gene Number

Gene 
number 6000 19,000 13,500 30,000 30,000
Simple Calculation
Human beings have roughly 3 billion base pairs
of DNA in 23 pairs of chromosomes
Distance between bases is 3.4 Angstrom
For human this would be 3.4x3x109
Angstroms. or 1.02 meters per haploid
genome,
2.04size
The meters pernucleus
of the cell is roughly 10 micrometers
in diameter
The average size of condensed mitotic human
chromosomes is ~ 1 micrometer (>10,000 fold)
If there is no compaction, nucleus would be too small
To hold all DNA!!!
Function
• Storage of genetic information
• Precise segregation of replicated DNA into two
daughter cells
• Platform for transcription, replication, recombination
and DNA repair

Problem
How to retrieve genetic information from DNA
packaged into chromosomes?
How the long linear DNA molecules are
packaged into compact chromosomes?
Historic View: Chromosomes and
Chromatin
1879 : Walter Flemming discovered chromosomes,
observing threadlike structures in the nuclei
of salamander cells during cell division
Early 1900s, cytologists Walter Sutton and
Theodor Boveri, independently published
papers linking chromosomes to the Mendelian
principles of segregation and independent
assortment.
Their work inspired the chromosomal theory
of inheritance

This theory states that hereditary information is on


genes and that genes are located on 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.
Chromosomes are typically stained by dyes that
distinguish between areas rich in A-T nucleotide
pairs and areas rich in
C-G pairs. This
results in a pattern of
banding that is unique
to each chromosome.
Cytogeneticists use
these to detect major
chromosomal
centromere
abnormalities.

large rRNA
Compaction of chromatin is cell-stage dependent
A. Interphase chromatin B. a mitotic chromosome,
which is duplicated already

Question: How this compaction is achieved?


Compaction of chromatin
Nucleosome is the repeating unit of chromatin
 1974

A) 30 nm fibers
B) beads on a string­nucleosome
From interphase nucleus
Composition of Chromatin

DNA
Stable association
Histones (H2A, H2B, H3, H4 and linker histones)

Non­histone chromosomal proteins
In general not as stable as DNA-histone interactions
Nomenclature
Nucleosome = a nucleosome core particle + linker
DNA+ a linker histone
DNA length: 180-200 bp
Nucleosome core particle = histone octamer + 146 bp DNA
Nucleosomes can be 
isolated by digesting
with nucleases that cut
between the nucleosomes
in a region called the linker
Nucleosome­octamer
2 each H2A, H2B, H3, H4

142 hydrogen bonds between DNA and nucleosome,
mostly between phosphodiester bonds and amino acid
backbone of histones
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
Histones
– folding and coiling chromosomes
– 45% of the total mass
– 60 million molecules of each type per cell
Non-histone proteins (NHPs, acidic
proteins, nonhistone chromosomal
proteins, NHC proteins)
– help regulate DNA transcription and
replication
– at least 30 types
Histone fold­
3 alpha helices
 and 2 folds
N terminal tails are
subject to covalent
modification­important
for transcription
Histone self­assembly
The position of the core histone in the
nucleosomes
Nucleosome core particle
2.8 A crystal structure of the
Mono-nucleosome From Luger et al
Nature 389: 251 - 260 (1997);
Histone tail interactions with DNA
From Luger et al
Nature 389: 251 - 260 (1997);
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
Nucleosome Positioning
1. Nucleosome positioning is not random and is defined
by translational positioning and rotational positioning.
5S rRNA gene, MMTV LTR and Xenopus TRβA
gene promoter
2. The local influences of DNA rigidity and curvature will
affect the precise positioning of nucleosomes.
Histone octamer prefers binding to AT rich sequence
3. In most cases, nuclosome at given piece of DNA can
adopt multiple different positions
4. Nucleosome positioning affects access of transcription
factors and other proteins to DNA.
The position of nucleosome can allow or disallow the
binding of a transcription factor depending whether its
binding site is incorporated into the nucleosome or
exposed in the linker region
linker

Core particle

Translational positioning Rotational Positioning


How to determine translational positioning experimentally?

Partial Micrococcal Nuclease


Translational Positioning Digestion

Determining
180-200bp 146 bp sequence
How to determine rotational positioning experimentally?

DNaseI partial digestion

Hydroxyl radical cleavage

Preferential Cleavage Sites


In vitro reconstituted
nucleosome
End-labeled DNA +
Histone octamer
The effect of Nucleosome Positioning on Transcription
­30 +1
p62 AP1 CREB/ATF
TATA(a/t)A(a/t) YYAN(t/a)YY

SRF DRBP TATA box INR


CTF/NF1­like
SIF

Upstream regulatory elements Core elements


Highly varied Present in almost all promoters
Gene-specific Recognized by basal apparatus
Bound by regulatory proteins

Structure of RNA Polymerase II Promoter


The assembly of DNA into nucleosome has
differential effect on binding of transcription
factors

Sensitive to nucleosome structure:


NF1, HSF, TFIIIA, TBP and etc.

Not sensitive to nucleosome structure:


Gal4, GAGA, thyroid hormone receptor and etc

This effect is dependent on nucleosome positioning!


How “the beads on a string” chromatin is folded further
into 30 nm chromatin fiber?
Linker histones in higher
order chromatin compaction
Histone H1 monomers link nucleosomes
Nucleosomes are further packed into a 30 nm fiber
the zigzag model
The 30 nm nucleoprotein
fiber
Solenoid Model - six to
eight nucleosomes per turn
Most interphase
chromatin is condensed
into 30nm coils.

Histone H1 helps this compaction but is not essential!


Further Compaction?
Chromatin in the interphase
nucleus is next believed to
organized into discrete
domains defined by sites of
attachment to the nuclear
matrix.
Interphase

M phase
Chromosome
scaffold

topoisomerase
Histone depleted
metaphase chromosomes
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
The state of condensation of chromosomes
varies according to the cell growth cycle. The
mitotic chromosomes are highly condensed, in
contrast to that of the interphase chromosome.
What structures are necessary to a
functional eukaryotic chromosome?
• Centromeres
• Telomeres
• Origins of replication
Three types of specialized sequences found in all eucaryotic
chromosomes ensure that chromosomes replicate efficiently.

many, to
ensure speed

kinetochore =
protein
complex that
binds the
spindle and the
centromere

The condensed state is important,


allowing the duplicated chromosomes to
be separated
Origins of Replication
Centromere
The Functions of Centromeres
The centromere is a highly differentiated structure of the
chromosome that fulfils a multitude of essential mitotic and
meiotic functions

•Required for chromosome stability

•Sister chromatid pairing

•Mitotic and meiotic spindle attachment

•Chromosome movement

•Cell cycle checkpoint control


In most cells, centromeres of mitotic chromosomes that have not yet
attained a stable bipolar orientation on the spindle would send a signal
to delay the onset of metaphase/anaphase transition
How do Centromeres Work?
site of kinetochore formation allowing attachment of
the sister chromatids to microtubules emanating
from each pole of the spindle
kinetochore = large protein complex mediating spindle
attachment to chromosomes
The Structure of Centromeres
• One centromere for each chromosome
• Structurally complex
• Kinetochore - spindle fiber attachment
• required for the stability of chromosomes
during mitosis
• DNA at yeast centromeres is relatively simple
• human centromeres 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 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!
B. Telomeres
Telomeres:
•allow complete replication of the ends
of chromosomes
•protect them from erosion and fusion
with other DNA fragments.
Cell prepares for mitosis Mitosis:
makes essential proteins division of 1 complete
set of chromosomes
to each progeny nuclei

• prepare for DNA


replication
• gets large enough to divide
• most of time spent in this
• Duplicates DNA phase
• Identical sister chromatids
• joined at centromere
Interphase Nucleus: euchromatin vs
heterochromatin
Interphase chromatin has more- or
less-condensed states, depending on how
“active” it is, I.e. are the genes being transcribed?
A. Euchromatin.
– open, dispersed, potentially active
– located at the interior of the nucleus
– Chromosomes have predictable and unique locations in the
nucleus.
B. Heterochromatin. during interphase about 10% of the chromatin
remains in a compact state similar to the mitotic chromosome.
– located at the periphery of the nucleus, looks darker
– this DNA is not transcribed or translated

66
– there are two types of heterochromatin:
1. Constitutive heterochromatin, condensed at all times
– includes AT-rich satellite DNA at the centromere
– the telomeres, or ends of the chromosomes (especially in
plants)
2. facultative heterochromatin transient condensation,
contains potentially active genes
– e.g. less active chromosome: chromosome 18
– e.g. one X chromosome (from mother or father) is
“turned off” early in development
» this becomes a “Barr body”
» in subsequent daughter cells, the same chromosome
stays condensed
» eventually the Barr body does get reactivated when
new germ cells are made
– facultative heterochromatin becomes more abundant in
cells as the organism matures from embryo to adult, as
cells specialize fewer genes are active
22.228 lecutre 7 67
Lampbrush and polytene chromosomes
Lampbrush chromosomes: found in the oocytes of many animals.
They contain very transcriptionally active DNA, where loops of DNA
emerging from an apparently continuous chromosomal axis are coated
with RNA polymerase. Each RNA polymerase is attached to nascent
RNA and associated proeins generating a visible ‘brush-like’
appearance.

The transcription of the RNA precursor of the 28S, 18S, and 5.8S ribosomal RNAs. These
units are tandemly linked together, some 450 per haploid genome.
Polytene Chromosomes of Drosophila

Polytene chromosomes are


“giant” chromosomes that
have undergone many
rounds of DNA duplication
without cell division.

Their large size makes them


easily visible under the
compound light microscope,
and makes them amenable for
various genetic studies.