Ch. 19 Gene Regulation in Eukaryotes

Differential Gene Expression

Elements within DNA (Cis-acting)

Factors outside of DNA (Trans-acting)

Eukaryotes level of complexity (4)

1. Complex chromatin structure

2. RNA processing events

3. Transcription takes place in the nucleus and translation takes place in the cytoplasm

4. Gene regulation controls cellular differentiation into hundreds of specialized cell types

Steps involved in eukaryotic gene expression (6)

1. Transcription initiation

2. Transcript processing

3. Export from nucleus

4. Translation of mRNA

5. Protein localization

6. Protein modification

Cis-acting regulatory elements

elements of DNA near the gene of interest that control transcription

• EX: Promoters, Enhancers, Insulators

Trans-acting factors

proteins that interact with cis-elements to control transcription initiation

• Types: Transcription factors (basal factors, activators or repressors)

Promoters

A major cis-acting DNA sequence located directly adjacent to a gene

• binds RNA polymerase, often contains a TATA box , and allows a basal (low) level of transcription

Enhancer

major cis-acting DNA sequence that can be located far away from a gene (either 5' or 3' to the transcription start site)

• increase or decrease the basal level of transcription, functioning regardless of its position relative to the promote

Reporter Genes (for identifying enhancers)

constructs a recombinant DNA molecule that fuses a putative enhancer sequence to a reporter gene (such as GFP), and then generating a transgenic organism with that recombinant DNA in its genome

• EX: transgenic mouse expresses GFP in eye

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing)

Transposase inserts sequence adaptors ("tagmentation") into open chromatin regions, which are mapped via peak calling to identify possible enhancer

• EX: intact vs regenerating limb reveals active enhancers

How to validate ATAC-seq peak (i.e AmPeak7) as enhancer

fuse the candidate DNA sequence to a reporter gene (such as GFP), inject it into embryos to generate transgenic animals, and observe whether the reporter activates in the target tissue or condition (such as upon limb loss)

VISTA Enhancer Browser

catalogue of experimentally validated human and mouse noncoding fragments with gene enhancer activity

Enhancers in Shh gene

fine-tune gene expression (such as controlling Shh in developing limbs) and can be highly conserved across species

• allows genes in lungfish to function in mouse

Transcription factors

sequence-specific DNA-binding proteins that bind to promoters and enhancers and recruit other proteins to influence transcription

• Types: basal, activators, repressors

Pathway of assembly at promoter

TBP binds to TATA box → TAFs bind to TBP → RNA pol 2 binds to TAFs

Mediator

doesn’t bind DNA directly – bridges RNA pol II at the promoter and activator or repressor proteins at the enhancer

• 20+ proteins

Activators (on transcription)

Stim recruitment of basal factors + RNA pol 2 to promoters & recruit coactivators to open chromatin structure

DNA binding domains

specialized structural region of an activator protein that fits into the major groove of DNA to recognize and bind specific sequences

• EX: zinc finger or helix-turn-helix

Dimerization domain

specialized enhancers for polypeptide-polypeptide interactions

• EX: leucine zippers

• Homodimer + Heterodimer

Homodimer vs Heterodimer

Multimertic proteins made of identical subunits vs nonidentical subunits

Copressor

protein recruited by a repressor bound to an enhancer that suppresses transcription initiation

• Prevent RNA pol 2 from binding to promoter

• Modify histones to close chromatin structure

How indirect repressor interferes with activator

1. Competition due to overlapping binding sites

2. Repressor binds to activation domain (quenching)

3. Binding to activator and keeping it in cytoplasm

4. Binding to activator and preventing homodimerization

Transcription of Drosophila Dorsal in diff cells

recruits a coactivator and activates Gene 1 → corepressor Groucho, turns into a repressor of Gene 2

How do ~2,000 transcriptional regulatory proteins fine-tune the expression genes across different tissues?

Each regulatory protein can act on many genes + each enhancer has binding sites with varying affinities for activators and repressors

Insulators

DNA sequence located between an enhancer and a promoter that binds CTCF proteins to block enhancer access and establish topological domain boundaries

Polydactyly

due to deletion of TAD boundary between EPH4A and IHH genes = altered gene expression = extra limb/finger