CR3 Flashcards
The authors utilized a staining method referred to as TUNEL to detect apoptotic cells. What is the basis for TUNEL staining and how does it discriminate apoptotic cells?
In a cell that’s dying, the double stranded DNA is breaking and nicks are formed. TUNEL which stands for TdT (terminal deoxynucltotidyl transferase) dUTP nick end labeling assay uses TdT to repair these breaks. TdT will bind to the double stranded breaks in the dying DNA, it will then make the break into a blunt end. The TdT then starts adding dUTP to the terminal region of the DNA, this is template independent. This addition of the dUTP forms a loop. This means that TdT is often recruited during breakdown of DNA in the cell. If there’s breakdown of DNA that means the cell is undergoing apoptosis. Therefore, we can correlate apoptosis with the amount of TdT activity inside the cell. Now you can add a tag to the dUTP. This means that TDT can catalyze the attachment of deoxynucltotides tagged with a fluorochrome or another marker, to the 3’ terminal of DNA. Then you can measure how much the DNA fluoresces. You can tell the cell is going through apoptosis because there will be lots of breaks in the DNA and lots of TdT trying to repair the breaks and dUTP fluorescing.
) Why is caspase expression a marker for apoptosis? Describe one additional marker or indicator for apoptosis
Caspase expression is a marker for apoptosis because it is involved in prompting the changes detected when cell is undergoing death. It is an important marker of cells entry into apoptotic signaling pathway. In the experiment, it confirmed that only the myoblasts depleted for BrgI had increased levels of cell death. Caspase-3 was shown to have activated as early as one day after infection with the Ade-Cre virus. This suggested the expression of BrgI which is necessary for proliferation, reduced soon after infection by the virus leading to the increased activation of the caspase enzymes for apoptotic pathway. In addition, rescued BrgI deficient myoblasts from apoptosis were indicated by a decrease in the expression of activated capase-3. Additional maker or indicator for apoptosis includes lamins that make up the inner lining of the nuclear envelop. Cleavage of lamins leads to the dismantle of the nuclear envelop and causes the nucleus to shrink, thus, cell death.
In the ChIP experiment (Figure 5) the authors utilize IgH as a “specificity control”. What is meant by this term and why is it a critical control for the experiment?
They used IgH as a specificity control because Brg1 binding was not observed at the specific sequence they used in figure 5a compared to the IgG control which showed binding to Brg. This was done to show whether or not Brg1 controls chromatin remodeling at the pax7 promoter.
What is the purpose of the restriction enzyme accessibility assay (REAA)? Explain your answer in relation to the role of the chromatin remodeling process at the Pax7 promoter
The purpose of the REAA was to analyze the binding of Brg1 to the Pax7 promoter sites that was indicated in the previous ChIP experiment results.
It was shown that the depletion of Brg1 upon Ad-Cre infection resulted in a decreased nuclease accessibility at Pax7 promoter Pvull cleavage sites. This indicates that Brg1 was responsible for remodeling the chromatin structure at those areas to improve the nuclease accessibility, as once depleted, chromatin became dense and inaccessible.
Using four primer sets located at different areas of the Pax7 gene, the increase in accessibility was found to affect the promoter site present in primers 1 and 2 (347bp, and 637bp upstream). Chromatin accessibility was unaffected in primers 3 and 4 (6kb upstream, and inside the transcribed region). This data suggests that Brg1 functions as a co-activator at the Pax7 promoter, as a subunit of the chromatin remodeling SWI/SNF complex
Which two promoter sites are identified as targets of Brg1 binding? Which two promoter sites are not identified as targets of Brg1 binding? What does the differential binding of Brg1 to the promoter say about its role in activation of the Pax7 promoter?
In order to identify where BrgI controls chromatin remodeling at Pax 7, cleavage sites in the promoter region were identified. It was determined that Pax 7 contains several cleavage sites in the promoter region of pax 7.The two promoter sites that are targets of Brg1 binding 347 and 637bp upstream of the transcription site of the promoter region of pax. Pvull sites located within 6kb upstream if the pax 7 transcription site or within the pax 7 coding region showed no change in chromatin accessibility in any of the cells tested thus were not identified as targets of Brg1 binding. A reduction in nuclease accessibility at the Pax7 promoter by depletion of Brg1 by Ade-Cre infection resulted in decreased levels of Pvull cleavage. This proposes a model where BrgI as the enzymatic subunit of the chromatin remodeling SWI/SNF complex, functions as a co-activator at the pax promoter in proliferating myoblasts to drive pax 7 expression.
Brg1 may be a co-activator for C/EBPß transcriptional activator protein. C/EBPß is a direct regulator of Pax7 expression. Both Pax7 and C/EBPß are down regulated upon differentiation signaling. A binding site for C/EBPß was found in Pax7, and C/EBPß also stimulated the expression of Pax7 reporter genes. Brg1 has also been shown to co-activate C/EBPß. This could mean that Brg1 is dependent of C/EBPß for Pax7 transcription. Y5
The results of this experiment suggest that Brg1 ATPase associates with the Pax7 promoter. Describe an experimental approach that would also point to the direct involvement of Brg1 with the Pax7 promoter
To analyze the association of Brg1 ATPase with the Pax7 promoter, a DNA electrophoretic mobility shift assay (EMSA) could be performed. EMSA is used to study the degree of affinity or specificity of proteins to DNA oligonucleotides. This technique features polyacrylamide or agarose gel electrophoresis to examine the mobility of the substrate. Analysis is based upon the understanding that DNA protein complexes will migrate more slowly than their individual complexes, and is often termed a gel retardation assay as well. With the addition of Brg1 and associated protein targeting antibodies, protein identity can be confirmed through the formation of even slower antibody-protein-DNA complexes. Given the Pax7 promoter sequence, Brg1 and an antibody that targets Brg1, the specificity of the interaction could be properly ensured.
