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)

Folding of DNA into chromatin

Horn & Peterson Science 297:1824-27

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

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 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)

5-methyl-C and MutationsCaused 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 notrapidly removed.