CH 10 Epigenetics

Waddington’s genetic landscape

Cell fate during embryonic development

genetics vs epigenetics

Genetics fixed and irreversible

Epigenetics: non DNa sequence based heritable changes to gene expression

Dynamic and reversable

ENCODE PROJECT

determines functional elements across the genome

Transcirptome (RNA SEQ)

Epigenome (CHip-seq)

Debates regarding if definition of funcitonal

Chromatin

Mixture of DNA, Protein, and RNA

organize genome

control gene expression

Carrier of epigenetic information

Nucleosomes

Octameres of histones with 146 bp of DNA wrapped around 1.65 turns of a flat left handed superhelix

STRING oF BEADS

Euchromatin

light packed, gene rich

areas for active transcirption

Heterochromatin

Tightly packed

Mostly satellite sequences

Constitutive0 repetitive DNA, gene poor, later plicating,

Facultative0 more selectively inactivated cell type specific, more easily reversible

Chromatin modifying enzymes

Add/remove chemical groups, such as methyl phosphate, acetyl, and ubiquitin, to DNA or histone tails

Chromatin remodeling complexes

Uses ATPase activity to catalyze nucleosome mobility

Writer

DNA methyltransferases (DNMTs)

Histone acetyl transferases (HATS)

Histone methyl transferases (KMTS(

Eraser

Histone deacetylases (HDACs)

Histone demethylases (KDMS)

Chromatin remodelers

Switch/sucrose non fermentable

Open up chromatin for transcription

DNA methylation

Happens at dinucleotide CPG

CPG methylation

associated with gene silencing

heterochromatin thats methylated

promoters of genes in euchromatin that are not methylated

Recognized by methyl cpG binding domain containing proteins for transcription for repression

5‐Methylcytosine is mutagenic (Me-C to T),

thus, CpG’s are rare in the genome

Methylome – Bisulfite sequencing

Bisulfite converts C to U, but not 5meC

• Amplified DNA have T at unmethylated C,

C at methylated C

• Epigenetic -> Genetic change

• Sequence treated/untreated

samples to distinguish C/5mC

histone modifications

methylation

acetylation

phosphorylation

ubiquitarian

Histone code

Patterns of histone modifications

• Affect chromatin accessibility (nucleosome

structure/positioning)

• Affect what factors bind to chromatin

(Readers and writers of the histone code)

• “The combination of all histone

modifications within a genomic locus (input)

determines the flow of genetic information

arising from this site (biological output)”

Primary histone modification

Methylation

• Acetylation

Minor histone modification

Phosphorylation

• H3S10P: enriched on mitotic chromosomes.

• Marker for cells undergoing mitosis

• Mono-ubiquitination

• ubH2B at K120: active transcription

• ubH2A at K119: inactive transcription

Histone methylation marks

H3K9me2 or H3K9me3:

• constitutive heterochromatin

• e.g., repeat DNA, centromeres, telomeres

• H3K4me3: euchromatin

• H3K27me3: facultative and constitutive

heterochromatin

• H3K36me3: euchromatin

Histone acetylation

• Usually mark euchromatin region

• H3K9, H3K27, and H3K36 are targets of

methylation and acetylation.

• Methyl mark and acetyl mark can not be

deposited at the same lysine position on

the same molecule.

• So methylation can block

histone acetylation at the

same site.

Bivalency chromatin modification

• Two chromatin modifications in the same

region.

Histone modification assay

• Chromatin Immuno Precipitation

(ChIP)

• Isolate chromatin

• Bind antibodies specific to a

histone modification for isolation

• Sequencing to identify genomic

regions (ChIP-Seq)

C. Ku, N Naidoo, M Wu, R. Soong (2011)

Chromatin-remodeling

Important for transcription

• Transcriptionally active gene promoters possess

a nucleosome-free region

• Nucleosome positioning is regulated by ATP-

dependent chromatin remodelers (SWI/SNF).

Transcriptionally active gene

Transcriptionally silence gene

Chromatin accessibility assays

Nucleosome positioning:

• MNase-seq: Micrococcal nuclease

• cleave the linker regions between nucleosomes

• ATAC-seq: Assay for Transposase-Accessible Chromatin

• insert in the open linker regions

• Open chromatin:

• DNase-seq: DNase I hypersensitive sites

• ATAC-seq

M Tsompana and M Buck 2014

Actively transcribed genes

In open euchromatin

Associated with histone acetylation

(H3/H4Kac), tri-methylation of h3 lysine

*H3k4me3 at promoters and H36kme3) Over gene body

silenced genes

In densely packed heterochromatin marked by

DNA methylation (5mC) and H3K9me3

• In silenced polycomb domains marked by

H3K27me3.

Epigenetic vs genetic mutations

epimutations> changes in epigenome

Genetic mutations disrupt function

Epigenetic mutations typically misrelates gene expression through altering the chromatin context of the locus

Levels of gene expression control

1. DNA cis-acting sequence element

(promoter, enhancer)

2. 3-dimensional conformation of chromatin

3. Chromatin state (open, close)

4. DNA epigenetic modifications

5. RNA process (splicing, stability)

6. Protein Translation

7. Protein post-translational modification