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Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
Chromosome Structure:Essential elements
Chromosome 9 during mitosis
Centromeres:- involved in mitotic spindle formation- required for segregation of sister chromatids to daughter cells
Telomeres:-located at each end of each chromosome-required for completon of DNA replication
Origins of DNA replication:- required for the start of DNA replication- many origins per chromosome
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 strandedDNA (146 bp) wrapped around histone octamer
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.
Histone termini are extended
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)
Packing of chromatin & transcriptional activity
Horn & Peterson Science 297:1824-27
Less compactMore active
Highly compactinactive
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
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 modificationacetylationmethylation
phosphorylationother modifications
- Chromatin remodeling complexes
- Non-histone proteins - DNA modification
methylation of C at CpG
(Lecture topics)
(Lecture topic)
Histone ModificationAcetylation
•Histone acetylation leads to a loose chromatin structure.
•Transcription factors that stimulate transcription of genes bind histone acetylases.
Activated transcription factor
Histone acetylase
AcAc
Ac
Transcription
Condensed chromatin
Decondensed chromatin
Histone acetylation
Histone ModificationAcetylation
•Transcription factors that repress transcription of genes bind histone deacetlyases.
Activated repressor
Histone deacetylase
AcAc
Ac
NoTranscription
Condensed chromatin
Decondensed chromatin
AcAc
AcAcAc
Ac
Histone ModificationMethylation
•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)
Histone Modification
Maintaining the undifferentiated state of embryonic stem cells.
Genes with methylated H3K27 (not transcribed)
Genes with unmethylated H3K27 (are transcribed)
Lee et al (2006) Cell 125: 301-313
Genes required for
differentiation
Methylation of histone H3 lysine 27 (H3K27)
Genes required for
proliferation
Histone ModificationMethylation in Embryonic Stem Cells
•Histone H3 methylation patterns:
H3K27me(condensed chromatin; repressed transcription)
H3K27me/H3K4me(condensed chromatin; minimal transcription)
H3K4me(decondensed chromatin;
active transcription)
Bernstein et al (2006) Cell 125:315-326
K27 - lysine 27K4 - lysine 4
Histone ModificationMethylation in Embryonic Development
H3K27me
H3K27me/H3K4me
H3K4me
Bernstein et al (2006) Cell 125:315-326
Typical methylation pattern of key regulatory genes in undifferentiated embryonic stem cells:
Differentiation
Methylation pattern of genes that are repressed
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 ModificationMaintaining the methylation state
(a hypothesis)Trojer & Reinberg (2006) 125:213-217
H3K27 methylase PC binds to histone H3
amino tails
DNA MethylationIn mammalian cells, DNA methylation is associated withchromatin inactivity. DNA is methylated on the 5 position of C at some CG sequences.
...CG... ...C5mG...
Nucleotide:
Sequence:
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...
...CmG ...
...G Cm... +...CmG ......G Cm...
DNAreplication
CmG specificmethylation
Parent cell Newly replicated DNA Daughter cells(both strands (one strand (both strandsmethylated) methylated) methylated)