Epigenetics Theory

Quiz 2

How are all the different cells in your body produced from a single genome?

• Zygote → 200+ types of cells in human body

• Cells vary in phenotype due to unique gene (and thus protein) expression profiles

How are all the different cells in your body produced from a single genome?

• Gene expression is regulated by both extrinsic and intrinsic cues

  • Genes may be turned ‘on’ or ‘off’, or transcript levels can be increased/decreased

• extrinsic ←> intrinsic (bidirectional relationship)

gene expression is regulated by

both extrinsic and intrinsic cues

• Genes may be turned ‘on’ or ‘off’, or transcript levels can be increased/decreased

extrinsic

from the cell’s environment

• other cells

• organism's environment

  • E.g., growth factors trigger intracellular signaling cascades → changes in transcription

intrinsic

• DNA modification

  • Cell’s own machinery chemically modifies DNA in a way that affects gene expression

What is epigenetics?

• Study of changes in gene expression (and thus phenotype) that occur due to chemical modifications of the genome, rather than a change to the DNA sequence itself

  • Cells have the mechanisms to copy epigenetic modifications during divisions

  • These modifications are however, reversible

What is epigenetics? - human twins

• Lack of concordance in patterns of disease

  • 30 – 60% concordance rate for vast array of diseases such as: schizophrenia, AD, MS, Crohn disease, asthma, diabetes, prostrate cancer

    • Only 10% concordance rate for breast cancer

What is epigenetics? - other

• Phenotypic variations can arise in absence of changes to the nucleotide sequence of genes

• Experience and the environment can promote or inhibit gene expression

• A lifetime of experience can alter behaviour, via the epigenome

What is gene expression?

Process by which cells convert the information encoded in our DNA into a functional product

Gene structure and expression

• Genes are made of DNA, and most genes code for protein products

  • Consist of coding regions and noncoding regions

  • Transcription initiated at the promoter region

DNA consists of two

polynucleotide chains

• template strand

• coding strand

Template (-) strand (aka antisense strand)

• Read by RNA polymerase (RNA pol II)

• aka noncoding strand

Coding (+) strand (aka sense strand)

Sequence of mRNA is identical to this strand

Control of transcription in eukaryocytes

• Each cell in the body contains the DNA for every gene, but only expresses a subset of genes as RNAs

  • The brain expresses more genes than any other organ

  • Diverse populations of neurons have different gene expression profiles

• ‘Upstream’ regulatory regions

• Unique complement of transcription factors interacts with promoters and enhancer

‘upstream’ regulatory regions

Promoters, enhancers, and silencers ensure that the right gene is expressed in the right cells, at the right time

Unique complement of transcription factors interacts with

promoters and enhancer and silencers

Control of transcription in eukaryocytes

• Proximal regulatory region: Promoter

• Distal regulatory region: enhancers and silencers

• Promoter consists of a ‘core’ rate promoter region and ‘promoter proximal’ regions

  • Core promoter contains the TATA box

• Distal regulatory regions are specific to particular genes and particular tissues

Transcription initation in eukaryocytes

Regulatory regions are recognized and bound by protein complexes

How can special transcription factors attached to distal regulatory regions affect the core promoter and its complex of basal transcription factors?

• Special transcription factors called activators and

  repressors, bind thousands of bp upstream of transcription

• *control expression of specific genes

• Activators – help basal TFs and/or RNA polymerase bind to core promoter

• Repressors – may impede basal TFs or RNA polymerase such that they cannot bind to core promoter to initiate transcription

Ex) CRE binding proteins (CREBs) bind to the CRE sequence

• Special transcription factors often recognise short lengths of palindromic DNA

• cAMP response element (CRE site) = cAMP responsive element

  • 5’TGACGTCA3’ → ————AGT3’ usually palindrome sequence

• Phosphorylation of CREB allows CREB binding protein (CBP) to attach to CREB

activator/repressor or enhancer/silencer

Combinatorial regulation

• Genes may be controlled by several different transcription factors

• Certain combinations of activators may need to be present (in the absence of certain repressors) to induce gene expression

Cell-type specific gene expression profiles

• Different cell types express characteristic set of transcription factors

• Epigenetic modifications also determine whether a gene is turned ‘on’ or ‘off’

  • Important during development/cell differentiation

    • Genome-wide patterns of epigenetic modifications are established in early development

  • Over an organism’s lifetime, the environment influences the epigenome

How is DNA organized within the cell?

• 60 trillion cells, 3.2 billion base pairs (haploid genome); ~20 000 protein coding genes (Amaral et al., 2023)

  • ~2 m of DNA!

How is DNA organized within the cell?

DNA packaging step 1

Nucleosomes are the unit of chromatin

• DNA has a negatively charged phosphate backbone

• DNA wrapped around core histone octamer

  • H2A, H2B, H3, H4 proteins

  • H1 linker

  • 147 bp, wrapped 1.7 times

• Primary function of histone proteins is transcriptional control

DNA packaging step 2

30 nm chromatin fibers

• Transcriptionally dormant

• Linker histones and N-terminal tails facilitate interactions between nucleosomes into higher order structures

DNA packaging steps 3 and 4

300 nm fiber compaction in chromosomes

• 30 nm fiber forms loops anchored to a protein scaffold, establishing a 300 nm fiber

• 300 nm fiber further folds in on itself, forming a chromosome

Euchromatin

• looser/uncoiled, “open” state

• transcriptionally active

• ~10 nm fiber

• Function: permissive to transcription

• Interphase chromosomes take up entire nuclear space

Heterochromatin

• condensed, or “closed” state

• transcriptionally inactive

• predominates in non-coding regions

• 30 nm+

• Functions: transcriptional repression, genome stability

-any given chromosome can have

tighter or loosen regions

Chromatin remodeling complexes

Nucleosomes are dynamic

• Large protein complexes (ATPases)

• Cooperate with specific DNA-binding proteins to

  repress/activate gene expression

  • transcription

  • DNA replication

  • DNA repair

Chromatin remodeling complexes

1. Nucleosome assembly — promote gene silencing

2. Chromatin access — exposes binding sites for DNA binding protein

3. Nucleosome editing — replacing histone with a different variant

Chromatin remodeling complexes can render chromatin more

accessible to DNA-binding proteins

• access remodellers

• switch/sucrose non-fermentable (SWI/SNF) subfamily of remodellers

• Recognize histone modifications

  • Mobilize/unwrap or eject nucleosome/histones

  • Transcriptional apparatus is granted access

Chemical modifications of the nucleosome dynamically regulate transcription

• Posttranslational modification of histones

  • Acetylation of lysines (K)

  • Methylation of lysines (K) and

    arginines (R)

  • Phosphorylation of serines (S) and threonines (T)

  • Ubiquitination of lysines (K)

• DNA modification

  • Cytosine methylation

Histone acetylation and deacetylation

• Histone acetyltransferases (HATs) –acetyl group added to lysine (+) (HATs → gene activation)

  • N-terminal tails (preferred targets) for loosening up

  • Internal globular domains

• Acetylation destabilizes chromatin structure AND recruits chromatin remodelling enzymes

• Reversed by histone deacetylases (HDACs)

  • Acetyl group: -Ac

HATs vs HDACs

Methylation and demethylation of DNA

• Methyl groups added to cytosine nucleotides by DNA methyltransferases (DNMTs)

  • Especially targeted to CpG dinucleotides

•  Represses transcription

  • Prevents binding of transcription factors be its bulky

  • recruitment of methyl-CpG binding domain (MBD) proteins (repressors)

• CpG sites are spread throughout genome and are usually methylated

  • Exception is CpG islands long stretches of DNA

  • Majority of gene promoters reside within CpG islands → usually not methylated

• methylation indicated by: -Me

de novo methylation, methylation maintenance, active demethylation

Chromatin Remodelling – Summary

• Condensed chromatin inaccessible to transcriptional machinery

• Modulation of chromatin structure – 2 general mechanisms

  1. Posttranslational modification (PTM) of histone tails and DNA methylation

  2. ATP-dependent chromatin remodelling enzymes

• Histone code hypothesis: PTMs occur in complex patterns which are “read” by cellular machineries

  • Different combos at various amino acid residues may lead to either

  activation or repression of transcription

Modulation of chromatin structure – 2 general mechanisms

1. Posttranslational modification (PTM) of histone tails and DNA methylation

2. ATP-dependent chromatin remodelling enzymes

Histone code hypothesis

• PTMs occur in complex patterns which are “read” by cellular machineries

• Different combos at various amino acid residues may lead to either activation or repression of transcription