1. Hybridization
Diagnostic
tools
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression

2. 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

3. 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)

4. 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).

5. 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.

6. 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

7. 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)

8. Folding of DNA into chromatin
Horn & Peterson Science 297:

9. Packing of chromatin & transcriptional activity
Less compact
More active
Highly compact
inactive
Horn & Peterson Science 297:

10. 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

11. An insulator-protein complex
Gaszner & Felsenfeld (2006) Nat Rev Genet 7:

12. 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.

13. Important regulators of chromatin structure
- Histone modification
acetylation
methylation
phosphorylation
other modifications
- Chromatin remodeling complexes
- Non-histone proteins
- DNA modification
methylation of C at CpG
(Lecture topics)
(Lecture topic)

14. 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 Ac
Transcription Ac Ac
Activated transcription
factor
Histone acetylase
Histone acetylation

15. Histone Modification Acetylation
Transcription factors that repress transcription of genes bind histone deacetlyases.
Decondensed
chromatin
Condensed
chromatin No
Transcription Ac Ac Ac Ac Ac Ac Ac
Activated repressor Ac
Histone deacetylase Ac

16. Histone Modification Methylation
Methylation of some histone lysines condenses chromatin. Examples:
Histone H3 lysine 9
Histone H3 lysine 27
Histone H4 lysine 20
Methylation of some histone lysines decondenses chromatin. Examples:
Histone H3 lysine 4
Histone H3 lysine 36
Histone H3 lysine 79
(Lecture topic)
(Lecture topic)

17. 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:

18. Histone Modification Methylation in Embryonic Stem Cells
Bernstein et al (2006) Cell 125:
Histone H3 methylation patterns:
H3K27me
(condensed chromatin;
repressed transcription)
H3K27me/H3K4me
(condensed chromatin;
minimal transcription)
H3K4me
(decondensed chromatin;
active transcription)
K27 - lysine 27
K4 - lysine 4

19. Histone Modification Methylation in Embryonic Development
Bernstein et al (2006) Cell 125:
Typical methylation pattern of key regulatory genes in undifferentiated embryonic stem cells:
H3K27me/H3K4me
Differentiation
H3K27me
H3K4me
Methylation pattern of genes that are repressed
Methylathion pattern of
genes that are expressed

20. 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

21. Histone Modification Maintaining the methylation state (a hypothesis)
Trojer & Reinberg (2006) 125:
H3K27 methylase
PC binds to histone H3 amino tails

22. 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...

23. DNA Methylation and Epigenetic Inheritance
A specific pattern of DNA methylation can be transferred
to daughter cells:
...CmG ...
...G Cm... +
...CmG ...
...G C ... +
...C G ...
...G Cm...
DNA
replication
CmG specific
methylation
...CmG ...
...G Cm...
Parent cell Newly replicated DNA Daughter cells
(both strands (one strand (both strands
methylated) methylated) methylated)

24. 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.