Discussion Paper 5 Flashcards
Enhancer regions of genes contain binding sites for…
Transcription factors which are cell or tissue-specific
True or false: basal promoter regions contain binding sites for cell-specific TFs
False - not necessarily but some do. Most promoter regions have binding sites for more general TFs
Which retinal cells tend to continue to develop/be born postnatally?
Rods, bipolar cells, muller glia
Most of the retinal development focussed on in this paper occurs (pre-natally/post-natally)
Postnatally
What cell fate decision is being examined in this paper?
Rod vs Bipolar cell
As determined by previous studies, what occurs when Blimp1 (a transcription factor) is knocked out?
There is a loss of photoreceptors and a gain in bipolar cells
What is the underlying big picture question of this paper?
How does a GRN affect the bipolar cell vs rod cell fate decision?
What is the underlying question of figure 1A?
What are the elements which regulate Blimp1?
What is the significance of sequence conservation in figure 1A?
Conservation is a good indicator that that region of the gene has some essential role - especially looking for non-coding regions with high conservation = good sign of regulatory element
What is the experimental approach used to address the question in figure 1A?
“Promoter bashing”: chop the promoter region into bits to see which parts are essential for driving expression
What is the most important finding of 1c?
Using the 12kb region identified in figure 1A is sufficient to direct transcription of EGFP to the retina
What was the main technique used in this study to determine if constructs were directing expression to the retina?
In vivo electroporation
What is the model organism used in this study?
Mice
Why did the authors co-electroporate with CAG-LacZ?
This is a control to make sure the electroporation was working - LacZ = the dye, CAG = ubiquitous promoter
How long is a typical enhancer?
<500bp
What is being demonstrated by figures 1b and 1c?
Breaking up the 12kb region into smaller bits to find the necessary sequence which is required for retinal-specific targeting - identify a 108bp region which is sufficient and necessary to direct expression to the retina
What are the underlying questions of figure 2E-K? (2)
- What is the fate of the cells which are endogenously driven by the B108 enhancer?
- When does the B108 enhancer turn on?
Describe the experimental approach used to address the question in figure 2e-k
Fate mapping using cre-lox system to examine the fate of cells that express B108 (when cre present under promoter preceding cre, will cleave cells and turn on egfp)
Describe the most important findings of figure 2e-k
Most of the cells with the B108 promoter are rod cells - b108 sufficient to illuminate the photoreceptor cells
What conclusions can be made about the cells that express B108-Cre?
The B108 enhancer directs expression to postmitotic photoreceptors
If B108 was expressed in progenitor cells, what would we see in this figure?
Bipolar cells would also be expressing EGFP - because we know they go through a cell fate decision
What data in the paper supports the fact that B108-expressing cells are postmitotic?
Did EdU labelling: EdU labels cells which are replicating, and there were no EdU and B108-GFP+ cells
What is the underlying question of figure 3?
Is B108 required for PR development?
What is the experimental approach used to determine the requirement of the B108 enhancer?
Used CRISPR to remove the B108 promoter, then use cre-lox system to mark PR cells which have switched fate to BP in the absence of the B108 promoter, or mark BPs
