Transcript No Slide Title

Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
Chromosome Structure:
Essential elements
Origins of DNA replication:
- required for the start of
DNA replication
- many origins per chromosome
Telomeres:
-located at each end of
each chromosome
-required for completon
of DNA replication
Centromeres:
- involved in mitotic spindle formation
- required for segregation of sister
chromatids to daughter cells
Chromosome 9 during mitosis
Chromosome Structure:
Genes
• Genes make up a small portion of the
genome. A gene contains:
–
–
–
–
Transcriptional conrol elements
Introns
Coding regions in the form of exons
Translational control elements (if translated)
Chromosome Structure:
Intergenic Regons
• Most of the genome is intergenic DNA
– Transcriptionally silent
– Intergenic loci can be examined by molecular
tools: polymorphic markers such as STRs.
• Important in forensics.
• Important for finding disease genes.
• STRs are also present in some introns (and in some
cases, exons).
Chromatin:
The nucleosome
Histone Octamer: (2 each of histones 2a, 2b, 3 and 4)
Double stranded
DNA (146 bp)
wrapped around
histone octamer
Histone termini are extended
Chemical modification of the histones (primarily at the NH3
termini) leads to altered nucleosome-nucleosome interactions.
These alterations are important forthe regulation of gene
expression.
Chromatin:
10nm filament
Nucleosomes are spaced along the DNA
- There are about 50 bp of DNA between
each nucleosome.
- When extended, this structure looks like
“beads-on-a-string” when viewed by electron microscopy.
Nucleosomes
50 bp of DNA between
nucleosomes
Chromatin:
30nm filament
The 10nm filament can be further condensed,
to a 30nm filament.
Important effectors:
Histone H1addition
DNA methylation
Histone deacetylation
One possible structure
(other structures have been proposed)
Folding of DNA into chromatin
Horn & Peterson Science 297:1824-27
Packing of chromatin &
transcriptional activity
Less compact
More active
Highly compact
inactive
Horn & Peterson Science 297:1824-27
Chromatin Domains
• A particular Chromatin structure can extend
over a large domain.
• Domains are separated by DNA sequences
referred to as insulators
• Domains are loops of about 50 kb, anchored
to the nuclear substructure
• In mitotic chromosomes, the loops are
anchored to a protein core
An insulator-protein complex
Gaszner & Felsenfeld (2006) Nat Rev Genet 7:703-713
Functional states of Chromatin
• Repressed chromatin
– Contains DNA that will never be transcribed in a particular cell
line.
– Seen histologically as heterochromatin
• Inactive & Potentially active chromatin
– Contains DNA that is not transcribed, but may be in the future.
– Seen histologically as euchromatin
• Active chromatin
– Contains DNA that is being actively transcribed.
– Seen histologically as euchromatin
Important regulators of
chromatin structure
- Histone modification
acetylation
(Lecture topics)
methylation
phosphorylation
other modifications
- Chromatin remodeling complexes
- Non-histone proteins
- DNA modification
methylation of C at CpG (Lecture topic)
Histone Modification
Acetylation
•Histone acetylation leads to a loose chromatin
structure.
•Transcription factors that stimulate transcription of
genes bind histone acetylases.
Decondensed
chromatin
Condensed
chromatin
Activated
transcription
factor
Histone
acetylase
Ac
Ac
Ac
Histone
acetylation
Transcription
Histone Modification
Acetylation
•Transcription factors that repress transcription of
genes bind histone deacetlyases.
Decondensed
chromatin
Ac
Ac
Ac
Activated
repressor
Condensed
chromatin
Ac
Ac
Ac
Histone
deacetylase
Ac
Ac
Ac
No
Transcription
Histone Modification
Methylation
•Methylation of some histone lysines condenses
chromatin. Examples:
•Histone H3 lysine 9
•Histone H3 lysine 27 (Lecture topic)
•Histone H4 lysine 20
•Methylation of some histone lysines decondenses
chromatin. Examples:
•Histone H3 lysine 4 (Lecture topic)
•Histone H3 lysine 36
•Histone H3 lysine 79
Histone Modification
Methylation of histone H3 lysine 27 (H3K27)
Maintaining the undifferentiated state of embryonic stem cells.
Genes required
for
differentiation
Genes with methylated
H3K27 (not transcribed)
Genes required
for
proliferation
Genes with unmethylated
H3K27 (are transcribed)
Lee et al (2006) Cell 125: 301-313
Histone Modification
Methylation in Embryonic Stem Cells
Bernstein et al (2006) Cell 125:315-326
•Histone H3 methylation patterns:
H3K27me
H3K27me/H3K4me
(condensed chromatin;
repressed transcription)
H3K4me
(condensed chromatin;
minimal transcription)
(decondensed chromatin;
active transcription)
K27 - lysine 27
K4 - lysine 4
Histone Modification
Methylation in Embryonic Development
Bernstein et al (2006) Cell 125:315-326
Typical methylation pattern of key regulatory genes
in undifferentiated embryonic stem cells:
H3K27me/H3K4me
Differentiation
H3K27me
Methylation pattern of
genes that are repressed
H3K4me
Methylathion pattern of
genes that are expressed
Epigenetic inheritance
• Regions of chromatin can be silenced (become
permanently transcriptionally inactive).
• The pattern of silencing can be maintained and
passed to daughter cells, thereby defining a
differentiated cell type.
– Example: Methylation of histone H3 lysine 27.
– Example: Methylation of DNA at CpG
Histone Modification
Maintaining the methylation state
(a hypothesis)
Trojer & Reinberg (2006) 125:213-217
H3K27
methylase
PC binds to histone H3
amino tails
DNA Methylation
In mammalian cells, DNA methylation is associated with
chromatin inactivity. DNA is methylated on the 5 position of C
at some CG sequences.
Nucleotide:
Sequence:
...CG...
...C5mG...
DNA Methylation and Epigenetic
Inheritance
A specific pattern of DNA methylation can be transferred
to daughter cells:
...CmG ...
...G Cm...
DNA
replication
Parent cell
(both strands
methylated)
...CmG ...
...G C ...
+
...C G ...
...G Cm...
CmG specific
methylation
Newly replicated DNA
(one strand
methylated)
...CmG ...
...G Cm...
+
...CmG ...
...G Cm...
Daughter cells
(both strands
methylated)
5-methyl-C and Mutations
Caused by Deamination
If dC is deaminated,
dU is the product. dU
is rapidly removed by
DNA repair enzymes.
If d5-methylC is deaminated,
T is the product. It is not
rapidly removed.