Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
22 Cards in this Set
- Front
- Back
|
What are some myogenic regulatory factors? |
-MyoD -Myogenin -Myf5 -MRF4 |
|
|
What are model systems used to study skeletal muscle development? |
-C2C12 cells: mouse myoblasts -10T1/2: fibroblast cell line -primary myoblasts -knockout mouse models |
|
|
How were target genes of Pax7 found? |
-performed microarray analysis using C2C12 cells overexpressing Pax7-Flag *increase Pax7 led to increase in Myf5 |
|
|
How can you identify Pax7 interacting proteins? |
-Tandem affinity purification (TAP-Tag): 2 tags (His & Flag). Flag removed, then His, then Mass spec -control: Flag-His w/o protein -advantage: double purification step -disadvantge: takes a long time |
|
|
How does Pax7 regulate Myf5 expression? |
-induces chromatin modifications to promote transcriptional activation -binds Myf5 enhancer |
|
|
What regulatory complexes are recruited by Pax7?
|
-protein complex capable of methylating H3K4
(e.g. methyltransferase MLL2) |
|
|
How was it determined that Carm1 & Pax7 regulate Myf5 transcription? |
Knock-out cells or siRNA were used to determine in both Pax7 & Carm1 were needed for Myf5 expression |
|
|
What is the role of Carm1 in Myf5 expression? How was this determined? |
-Carm1 methylates Pax7 acting as a molecular switch controlling epigenetic induction of Myf5 -determined by methylation assay and use of Carm1 inhibitor |
|
|
How was MyoD discovered? |
-comparing cDNA libraries from 10T1/2, 5-Aza 10T1/2 and C2C12 cells. -forced expression of MyoD in non-muscle cell types can direct myogenesis |
|
|
What experiment could be used to show MyoD binding to E-boxes? |
-reporter assays -EMSA -mutate E-box sites and perform reporter assay to show binding is needed for transcription |
|
|
What are some issues with a E-box dependent model of skeletal muscle regulation? |
1. e-box motifs are found all over the genom 2. subfamilies of bHLH proteins recognize canonical sequences *more complex interaction, temporal specificity and promoter specificity need to be incorporated |
|
|
What experiment(s) was done to determine that MyoD forms a heterodimer with bHLH proteins? |
-EMSA: to show association of e-box proteins with MyoD -Radio-IP: radioactive e-box probe shows cna't bind w/o e-box proteins |
|
|
What experiment showed that coactivators p300 & pCAF (HATs) are needed for MyoD to initiate gene expression? |
in vitro transcription assay |
|
|
An experiment shows that Myogenin binds more genes in the presence of MyoD. What does this suggest? |
MyoD establishes an open chromatin state that allows binding of myogenin to permit efficient activation of muscle genes |
|
|
What are mechanisms of inhibiting MyoD activity? |
-block ability to bind e-boxes at target genes -block activation of transcription |
|
|
What are the HLH proteins that are antagonists of MyoD activity? |
-Id: expressed in proliferating myoblasts & down-regulated during differentiation -Twist -Mist-1 -Snail1/2 |
|
|
How does Id block muscle differentiation? |
-competes for MyoD/E-protein binding -Id bins e-protein, preventing e-protein from binding MyoD |
|
|
How does Twist block myogenesis/inhibit myoblast differentiation? |
1. dominant negative inhibitor by competing for e-proteins 2. binds e-box sequences that would normally be occupied by MyoD-E protein dimers, which shuts down transcription *limits boundaries of skeletal muscle development |
|
|
How does Mist-1 inhibit muscle gene expression? |
1. forms inactive heterodimers with MyoD 2. forms Mist1 homodimers which bind e-box sites & represses transcription 3. Mist1-Eprotein dimers bind e-box DNA but no transcription |
|
|
How does Snail1/2 repress muscle gene expression? |
1. Snail1/2 recruits HDACs and acts as transcriptional repressors. ChIP-seq revealed that Snail-HDAC bind to many differentiation-specific genes during myoblast growth 2. abolishes MyoD/E activity of G/C-rich E-boxes |
|
|
What are the preferential binding sites of MyoD through differentiation? |
-more A/T-rich e-boxes are utilized in myoblast enhancers -MyoD preferentially binds G/C-rich e-boxes in myotubes |
|
|
How is Snail1/2 removed at the onset of differentiation? |
Snail1/2 is a target of miRNAs |
