Lecture 5 Flashcards
(25 cards)
TFs have 3 domains
-DNA binding domain
-Trans-activation donation
-protein-protein interaction domain
How are a TFs organized?
Into families depending on how similar they are to each other in their DNA binding domains
What are pioneer TFs?
-bind to DNA that si vv tightly wrapped
-recruit histone modifying proteins to open up chromatin a bit
-which allows TFs to bind to enhancers/promoters
-can be tissue specific (FoxA1 -> liver)
How many pioneer TFs do you need for each normal TFs?
One pioneer Tf is enough to open up a marre area of heterochromatin.
-so each TF does not need a pioneer Tf since one pioneer TF can allow access to multiple enhancers.
Yamanaka factors
Shinya Yamanaka in 2006
-genes that maintain the undifferentiated state of the early mouse embryo: Oct3/4, c-Myc, Sox2, Klf4, etc.
-When these 4 genes are ectopically expressed in differentiated mouse fibroblasts, they dedifferentiate.
-These dedifferentiated cells are called iPSCs or induced pluripotent stem cells.
-You can then direct differentiation of these iPSCs into different cell types by expressing different genes and growing the cells in different conditions.
What is Gene regulation Network?
GRN
cascade of one TF activating another until last TF that will differentiate a cell
Pioneer TFs begin process of differentiation
What are the mechanisms of differential gene expression in pre-mRNA processing?
Alternative splicing
What is alternative splicing?
a process that allows for a single gene to form more than one protein
What is a spliceosome?
Splicing is accomplished by a complex called the spliceosome which is composed of proteins called splicing factors and small RNAs.
The spliceosome recognizes splice consensus sequences in the introns and catalyzes their removal.
What are the mechanisms of differential gene expression in mRNA translation?
-rate of translation
mRNA stability and translatability (4)
-half life
-stored oocyte mRNAs
-ribosomal selectivity
-microRNAs
microRNAs
How do microRNAs regulate translation?
through the destabilization of mRNAs and the blocking of translation
Where to miRNAs bind?
microRNAs bind complementary sites in the 3’UTRs of target genes
They often don’t bind with perfect complementarity (bulges in the middle)
What was the first miRNAs to be discovered? What does it do?
lin-4 was the first microRNA discovered. Discovered in C. elegans.
•lin-4 regulates a gene called lin-14. lin-14 has 7 binding sites for the lin-4 microRNA.
•The LIN-14 protein is a transcription factor that is only needed for the first phase of larval development, the mRNA needs to be quickly degraded after to stop production of the protein to allow the worm to proceed to the next larval stage.
•lin-4 is responsible for this degradation.
How does miRNA contribute to the maternal to zygotic transition?
Once fertilized, stored maternal mRNA starts translating
But we need to start stranslaring zygotic mRNA
M-to-Z switch -> degadation of maternal mRNA by miRNA, switch to zygotic mRNA
In zebra fish -> miR430 is responsible for around 40% of the degradation
Why is mRNA location important?
Because control of location of mRNAs mean control of location where proteins will be expressed
mRNA localization is controlled with 3 mechanisms
- Diffusion and local anchoring : protein can help bind mRNA in specific location
- Localized protection : specific mRNA degraded everywhere except specific location
- Active transport along cytoskeleton : most widely used, 3’UTR is recognized by proteins that bind mRNAs to motor proteins that travel along cytoskeleton to final destination
What are the mechanisms of differential gene expression in post-transitional regulation of gene expression? (3) how do they regulate?
They regulate how long proteins live within cell and what it does.
- Protein modification : Modifications can do anything from target a protein for degradation, alter its function, target it for localization to specific organelles
- Ligand binding : hemoglobin, ribosomes
- Protein stability
Protein modification examples (2)
Phosphorylation: enzymes, turn on and off
Glycosylation: secretion in certain membranes
Tools to study developmental biology (7)
-in situ hybridization
-CHIP sequencing (chromatin immunoprecipitation)
-RNA sequencing
-testing gene function
-reverse genetics
-CRISPR/CAS9
-The GAL4-UAS system
In situ
- Protease treatment (removing excess proteins)
- Probe hybridization (adding complimentary sequence to RNA)
- Adding enzyme (alkaline phosphatase) or fluorescent l’y labelled anti-bodies
- Adding substrate to see where reaction occurs
ChIP sequencing (chromatin immunoprecipitation sequencing)
To find binding site of protein of interest
- Formaldehyde (fix the proteins)
- Generate smaller DNA fragments (with sonic action or restriction enzymes)
- Immunoprecipitation with antibodies
- Purification
- Reversal of crosslinkage (heat or digestion)
- Sequencing
Helps to find DNA sequence bound by transcription factor of interest. This tells us which genes are activated by TF
RNA-sequencing
Objective: To identify the full repertoire of genes expressed in an embryo, tissue or even a single cell at a given moment.
The array of expressed mRNAs in a cell/tissue/embryo is called the transcriptome
The ligation of primers on the cDNA fragments is to allow a universal primer to be used for the sequencing reaction, instead of thousands of unique primers that are complementary to each fragment
testing gene function
To test a gene’s function, you have to prevent production of the gene’s protein product and assess how its loss impacts development.
•Two ways of doing this: forward genetics or reverse genetics
