Physiology 3140A Lecture Notes - Lecture 28: Cre Recombinase, Heat Shock Protein, Green Fluorescent Protein
2 May 2018
School
Department
Course
Professor
Cell Physiology Lecture 28
Epigenetics
December 4th 2017
Significance of Transcription Factors:
1) Essential mediators of developmental processes. Many developmental disorders are linked to
mutations or dysregulation of transcription factors
- Lim1?
o Transcription factor expressed during brain development
o 2 embryos at day 9.5
▪ Head is deformed when you knock out a single transcription factor – losing entire
brain and head region
- Mutations that are not common because you do not carry forward mutation that ended up with a
phenotype like this
2) Affecting the levels of transcription factors can also result in disease
- More subtle mutations:
o Transcription factor still made but LESS functional
o Mutate 1 of 2 copies – only have 50% of the transcription factor
- There is inheritance from one person to the other
o Families had occurrence of this type of diabetes
- Chromosomal area is linked to the progression of the diabetes. Identified the specific genes that were
mutated leading to the disease
o ALL except glucokinase are transcription factors
- All TF are needed for the gene. Loss of 1 TF causes disease (only 1 copy of the TF)
find more resources at oneclass.com
find more resources at oneclass.com
3) Development of research tools
o e.g. inducible systems for deleting genes in a tissue and spatial-restricted pattern
Clinical Purpose – Lox + Cre
- Nuclear receptors – common way to get rid of gene in temporal fashion, allowing the mouse to grow
and then specifically delete the gene
o Using nuclear receptor estrogen receptor
- Gene in nucleus & have a promoter always on
o LOX – LACZ- LOX P
o LacZ and EGFP are reporter genes
o Without any change to the gene setup, it will only express LacZ
▪ Only see blue when expressed due to stop signals preventing expression of EGFP
- Tissue specific promoter that is going to drive the CreERt
- Cre recombinase: bacterial enzyme specifically recognizes lox P sites & deletes everything in middle
o PROBLEM: if you just expressed the cre recombinase it will immediately go the nucleus and
take out that gene
- If you want a temporarily affect the loss of this area – fuse it to this estrogen receptor that is only
inducible by toamoxifen
o Binds to heat shock protein in the cytoplasm to keep cre recombinase in the cytoplasm
- Give NO tamoxifen: stays in cytoplasm
- Give tamoxifen: dissociates from heat shock protein, goes to the nucleus and cleaves the lox sites
causing expression of another gene
o Cells are now expressing EGFP
- THERAPY: Can drive expression of what you want in a tissue specific manner
Epigentics
- What is it? Heritable changes in gene expression that do not involve any change in DNA sequence
o Note: no mutations involved
o Way for environment to affect how genome is packaged = can lead to developmental
problems
o Heritable: one cell to another OR parent to offspring
find more resources at oneclass.com
find more resources at oneclass.com
- 3 general types
o 1) Modification of histone core proteins
▪ Could be phosphorylation, ubiquination, methylation, sumoylation and acetylation
• Can cause changes to chromatin where chromatin is unwound to allow
access to transcription factors OR make the area less accessible
▪ Can be associated with repression or activation
▪ Includes chromatin remodeling proteins
o 2) DNA methylation
▪ Generally, at cytosine residues
▪ Usually associated with gene repression
• Causes compaction of that area of the genome
o 2) MicroRNAs
▪ Affect transcription, silence genomic regions or alter RNA processing all leading to
changes in RNA accumulation and expression
▪ Short RNA come in & cause degradation of mRNA
▪ ALL NON CODING RNAS – 10 different classes working in different days
Histones
- Relatively small proteins, 100-300 amino acids which are mostly positive
o Interact with the negatively charged DNA
- DNA wraps around the histone bodies (made of a group of proteins/histones)
- Each subunit has an N-terminal tail that sticks out of the octomer core, these can be covalently
modified to change DNA wrapping
o Region that are targeted by the different epigenetic modifiers
- Each octomer is separated by a short piece of DNA called the linker DNA
o 8 proteins, but 4 dimers
- Nucloosome:
o DNA wraps around 2.5 times + linker region
- Linker DNA is couple hundred base pairs
o Region from one histone to where you wrap around the other histone is 200 bp
o SOMETIMES you space the nucleosomes further apart to access larger TF factor complexes;
linker region can be stretched
- Histones can be post translationally modified in several different ways
- Methylation/acetylation is the MOST COMMON epigenetic modification at the histone
- Methylation site determines what the impact is
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Significance of transcription factors: essential mediators of developmental processes. Many developmental disorders are linked to mutations or dysregulation of transcription factors. Lim1: transcription factor expressed during brain development, 2 embryos at day 9. 5, head is deformed when you knock out a single transcription factor losing entire brain and head region. Mutations that are not common because you do not carry forward mutation that ended up with a phenotype like this: affecting the levels of transcription factors can also result in disease. More subtle mutations: transcription factor still made but less functional, mutate 1 of 2 copies only have 50% of the transcription factor. There is inheritance from one person to the other: families had occurrence of this type of diabetes. Chromosomal area is linked to the progression of the diabetes. Identified the specific genes that were mutated leading to the disease: all except glucokinase are transcription factors. All tf are needed for the gene.
