W12L3 and W13L1 Flashcards
How can you regulate transcription factors?
Synthesis – Transcription/translation
- basically don’t make them
Access to DNA
Post-translational modifications
- phosphorylation can occur at multiple levels
Ligand binding
- triggers pathways causing phosphorylation
- can cause post-translational modifications: phosphorylation can occur at multiple levels
- At the surface, this leads to a signalling cascade that involves downstream mediators and post-translational modifications. e.g. TGFβ signaling, WNT signalling, Receptor Tyrosine Kinase signalling
Cellular localization
Availability of co-activators or co-repressors
Nuclear receptor - 2 pathways
- Steroid hormone binds to inhibitor complex within the cell cytoplasm
- when steroid bound to nuclear receptor, it releases from inhibitor complex and enters nucleus and can impact transcription - No inhibitor complex. Metabolite directly enters nucleus and binds to a complex that is sitting directly on the gene
- more efficient
- metabolite can activate or repress
Nuclear receptors
nuclear receptor superfamily is comprised of 48 genes (plus splice variants) in humans
Receptors shuttle to/or already present in the nucleus upon ligand interaction
regulate transcription by binding to specific DNA sequences
Can bind as monomers, homodimers or heterodimers
Nuclear receptor composition
N-terminalregulatory domain:
- Contains the activation function 1 (AF-1) whose action is independent of the presence of ligand.
- highly variable in sequence between various nuclear receptors.
DNA-binding domain(DBD):
- Highly conserved domain
- contains twozinc fingersthat binds to specific sequences of DNA calledhormone response elements(HRE).
Hinge region:
- connects the DBD with the LBD
- Influences intracellular trafficking and subcellular distribution
Ligand binding domain (LBD):
- highly conserved in structure between the nuclear receptors.
- Binds ligand leading to dimerization and coactivators/corepressors
- contains the activation function 2 (AF-2)
C-terminaldomain: Highly variable in sequence
Nuclear receptors classes
divided into four classes based on key characteristics such as dimerization, DNA binding motifs and specificity, and ligand binding
Classifications based on sequence alignments and DNA binding behavior
Class I: Steroid receptor (SR)
Class II: RXR Heterodimers
Class III: Dimeric orphan receptors
Class IV: Monomeric orphan receptors
Nuclear receptors – Class I
Localized to the cytoplasm
- Bound to an inhibitor such as Heat Shock Proteins (HSP)
Typically activated by hormones such as thyroid hormone, estrogen, androgens, glucocorticoids and mineralcorticoids
Bind as homodimers to activate gene expression
Roles in:
- maintenance of cellular homeostasis
- gene expression
- regulation in embryogenesis
- tissue development
binds hormone response elements (HREs) consisting of two half-sites separated by a variable length of DNA
second half-site has a sequence inverted from the first (inverted repeat)
Deletion of ERα or Erβ
Mice are viable but…
- leads to infertility
- CL, corpus luteum: evidence of ovulation and produces progesterone for early maturation of oocyte
- HC, hemorrhagic cyst
Nuclear receptors as research tools
inducible systems for deleting genes in a tissue and spatial-restricted pattern
Cre-recombinase targets specific DNA sequences (LoxP sites) and causes recombination
How do you make this cell or tissue specific?
- Express cre recombinase from a promotor that generates the specificity needed, allowing you to delete the gene only where the promotor is
HOW DO YOU MAKE THIS TEMPORALLY-SPECIFIC?
- Modified version of the estrogen receptor: estrogen receptor + cre recombinase + tissue specific
- it will bind to heat shock protein, causing ER to bind to heat shock protein
- and then cre recombinase will go to nucleus to splice out whatever is between LoxP sites
Overall:
- Express the modified creERT (estrogen receptive, tamoxifen-inducible) in a tissue specific fashion
- Deliver tamoxifen at the time required.
i.e. DCLK1 is only expressed in cells at the base of villi. LacZ+ cells don’t need to express DCLK1 AFTER recombination
Nuclear receptors – Class II
Localized at the gene
Bound to gene and maintains the gene in an off state
Typically activated by ligands and metabolites that can passively enter the cell and nucleus
Bind as heterodimers to activate gene expression, most commonly with Retinoid X receptor (RXR)
Roles in:
- gene expression
- regulation in embryogenesis
- tissue development
- Processing vitamins medicine and foreign substances
bind response elements consisting of two half-sites separated by a variable length of DNA
- Sites can be RARE (retinoic acid response elements), PPRE (PPAR responsive elements), etc.
- second half-site can be a direct repeat (DR), an inverted repeat (IR) or everted repeat (ER)
Involve switching genes from off to on states
Changes the affinity of the existing heterodimer from a co-repressor complex to a co-activator complex
Many of these complexes affect the packaging of DNA – i.e. epigenetic regulation
Nuclear receptor II in development
e.g. Retinoic Acid
metabolite of vitamin A that mediates the vitamin A functions required for proper development
Loss of retionic acid affects multiple developmental processes, like eye development and forelimbs are shorter or non existent
Retinol (vitamin A) to (via ADH or RDH/SDR) Retinal to (via Raldh) Retinoic Acid (Metabolized by Cyp26)
Nuclear receptors in cancer
- Affects inter cellular signalling and response to chemotherapy
also diabetes and obesity
Epigenetics
Things that control the expression of DNA that have nothing to do with the actual sequence of the DNA
Heritable from one cell to the next cell
Is transgenerational
- impacts embryo and embryo’s offspring
Definition: Heritable changes in gene expression that do not involve any change in DNA sequence
Three general types of epigenetics
- Modification of histone core proteins
– could be phosphorylation, ubiquination, methylation, sumoylation and acetylation
– can be associated with repression or activation
– includes chromatin remodeling proteins - DNA methylation
– generally at cytosine residues
– usually associated with gene repression - microRNAs
– affect transcription, silence genomic regions or alter RNA processing all leading to changes in RNA accumulation and expression
Pioneer factors
Going from heterochromatin to euchromatin, need to uncoil and unpack the histones using pioneer factors
Then use histone demethylase or demethylation to remove negative marks or remove methylation events that are repressive
Then add in markers that are positive, that will promote expression
Histones
Small proteins, 100-300 amino acids mostly positively charged
Subunits have N-terminal tails that sticks out of the octomer core, allow covalently modifications that change DNA wrapping
Nuclesomes are separated
by a short piece of linker DNA
Length of linker DNA can be
modified
Histones are translationally modified in many different ways
Acetylation (associated with transcription)
- histone acetyl transferases
Methylation (associated with expression or repression)
- Histone methyl transferases
Sumoylation (associated with gene silencing)
Phosphorylation (associated with expression)
Ubiquitination (associated with expression or repression)
Writers, readers, erasers
Writers
- put the epigenetic markers on
Readers
- recognized the modifications and carry out whatever is supposed to happen
Erasers
- remove the markers
Dutch Winter
start of November 1944 to the late spring of 1945
- People lived on 30% of their normal calorie intake
Cohort A: Pregnant mothers in the third trimester had that were smaller and remained smaller their entire life
Cohort B: Pregnant mothers in the first trimester had babies with a higher rate of obesity
- Children in cohort B showed increased risk for metabolic disease including diabetes, cardiovascular disease, and mental health
- Children of cohort B also showed increased risk for these diseases
Why was methylation affected by Dutch famine?
- Limited substrates for obtaining methyl groups in the diet; decreased intake leads to decreased availability
- after starvation, food arrives, and baby does catch-up growth. But by then, their programming had already changed, resulting in metabolic syndrome
Due to this study,
- folic acid is now provided as a supplement to pregnant women so that enough methylation is given to the fetus
Histone acetylation
catalyzed by histone acetyl transferases (HATs) – transfers an acetyl from acetyl-CoA to histone tails
HAT A enzymes – active only in the nucleus
HAT B enzymes – active in the cytoplasm and modifies new histones prior to incorporation into nucleosomes
Generally, acetylation causes DNA to be unwound to allow for transcription
Histone Deacetylases (HDACs)
remove acetyl group from histones
Class I HDACs (1-3, 8) are located only in the nucleus
Class II HDACs (HDAC4-7, 9, 10) are active in the nucleus and cytoplasm and shuttle between compartments
Unlike transcription factors, HATs and HDACs don’t directly bind DNA but are recruited by transcription factors
Histone Deacetylases (HDACs) in cancer
HDAC is involved in cancer steps: differentiation, proliferation, metastasis, angiogenesis, apoptosis, cell cycle, inflammation
Many HDAC specific inhibitors exist and are being tested in clinical trials but…
- poor bioavailability
- ineffective for solid tumours
- off-target toxicity
