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Ilona Skerjanc

1 BIOMACROMOLECULES REVIEW SKERJANC SECTION THINGS YOU NEED TO STUDY FOR PAPERS 1 4 1) You should be able to explain any figure in these papers that is shown to you, after being told the method used: What was done (no details in the method but should state basic experimental design and background info relevant to the experiment) What it means Was it done correctly? Was something left out? What could you do to improve the experiment? 2) You should be able to describe the overall model for each paper: Draw the model and explain it Explain which experiments supported this model (include the outcome of the experiment so it is clear how this experiment supported the model) Suggest other approaches or next step experiments 3) You should understand what each technique is used for and extrapolate this into new situations. PAPER #1: REGULATION OF SKELETAL MYOGENESIS BY ASSOCIATION OF THE MEF2 TRANSCRIPTION FACTOR WITH CLASS II HISTONE DEACETYLASES Lu et al., 2000 (Molecular Cell). An Erik Olson Paper QUESTIONS Do Class II HDACs regulate myogenesis? By what mechanism? How is the inhibition regulated? MEF2 and MRFs Act Cooperatively to Activate Muscle-Specific Genes Note: MRFs (myogenic regulatory factors) - MyoD, Myogenin, Myf6, MRF-4 - note adding MyoD to cell = myogenesis - MEF = myocyte enhancer factor - MEF2 = A/T rich sequence - MEF1 is an E box sequence that binds MyoD - bHLH = basic helix loop helix - Myogenic bHLH factors = activate muscle transcription as heterodimers with ubiquitous bHLH proteins known as E proteins and bind E boxes in regulatory regions of muscle target genes - these associate with MEF2 which binds a conserved A/T rich sequence in muscle gene regulatory regions MEF2C: - Is the first MEF2 to be expressed in developing skeletal muscle? - binds to HDAC by its MAD/MEF domain - binding of HDAC causes repression of MEF2C activity - the repression can be relieved by CaMK (calcium/calmodulin-dependent protein kinase) - calmoudulin is a ubiquitous Ca2+ binding protein - activates the serine/threonine kinase activity of CaMK - Note HDACS Class IIa (4 and 5) are expressed and enriched in heart, skeletal muscle, and brain - have both NLS (nuclear localization signal) and NES (signal export signal) HYPOTHESIS - Class II HDACs will inhibit myogenesis by binding MEF2C, in a CaMK-dependent fashion HDACS 4 AND 5 SPECIFICALLY INHIBIT MYOGENIC ACTIVITY OF MYOD (Figure 1) METHOD/EXPERIMENT USED MYOGENIC CONVERSION ASSAY 1) Transfected MyoD with/without HDACs into fibroblasts - DNA is taken up by eukaryotic cells when incorporated in a calcium phosphate precipitate - mix calcium phosphate and DNA, add to cells, and incubate 9 hours and then wash 2 2) Use immunofluorescence to visualize the cells - fix cells with methanol - incubate with primary antibody (anti-Myosin Heavy Chain) - incubate with secondary antibody (that is detectable under fluorescence) 3) MHC = skeletal muscle (differentiation) RESULTS FIGURE 1 HDACs 4 and 5 Specifically Inhibit Myogenic Activity of MyoD **figure 1A here** *** - % myogenic conversion where 100% = 50 cells/coverslip Therefore, HDACs 1 and 3 do not repress myogenesis, while HDACs 4 and 5 do. ** - Note that MyoD and HDAC are exogenous, MHC and assumed MEF2 are ENDOGENOUS - HDAC binds to endogenous MEF2, which interacts with MyoD (through yeast-2 hybrid and coimmunoprecipitation, no HDAC- MyoD binding) What domains of HDAC 4/5 are important for inhibiting MyoD? **here is figure with domains** - red band is the MEF2 binding site If MEF2 binding site is mutated, HDAC cannot bind MEF2 **Figures A and B here** - Repression of myogenesis by HDAC 5 was not seen in 1) the N-terminal deleted mutant of HDAC5 (HDAC5N), and 2) the HDAC mutant 175 192 (missing only the MEF2 binding domain). inhibition required MEF2 binding domain - Mutation of histidine 803 to leucine in HDAC4 in catalytic domain - if there is no catalytic activity, HDAC cannot deacetylate the histones and is not able to repress transcription - mutant severely impaired in ability to repress myogenesis, therefore ** inhibition requires MEF2-binding domain and functional catalytic domain ** - HDAC-VP16 = the fusion of Mef2 binding domain with VP16, a powerful transcription activation domain from a viral transcription factor. It can activate the function of many transcription factors - both HDAC4 and HDAC5 with VP16 - if transcription activator VP16 can be targeted to MEF2 by the MEF2 binding site, then we would expect transcriptional activation to occur - ** replacing the HDAC catalytic domain with VP16 resulted in an enhancement ** this data is consistent with the model in which HDAC inhibits myogenesis by binding MEF2, which binds to MyoD ** WHAT IS WRONG WITH THIS FIGURE? - There are no error bars!! ** - here they use representative experiment (which they supposedly did 3 times) DO HDACs CHANGE THE ACETYLATION STATUS OF MUSCLE-SPECIFIC GENES DURING MYOGENESIS? (FIGURE 2A) METHODS/EXPERIMENTS USED CHROMATIN IMMUNOPRECIPITATION (ChIP) - chromatin from cells of interest is isolated, cross-linked so proteins are linked to DNA, and then sheared. Antibodies are used to immunoprecipitate protein of choice (transcription factor of interest or modified histone). Cross-links are reversed, proteins are removed, and then the DNA they were attached to is purified. Purfied DNA is analyzed through PCR. SATELLITE CELLS Present in adult muscle Can be activated to proliferate and differentiate into skeletal muscle by injury Are the source of cells for various skeletal myoblast cell lines: C2 myoblasts - these satellite cells were used in the experiment - grown in growth medium (high serum) and then grown in differentiated medium (low serum) 3 - after MyoD expression during growth stage, you eventually get myogenin, and MCK expression during differentiated stage - what happens as cells go from myoblast stage to differentiated myotubes? HOW TO MAKE ANTIBODIES 1. Take blood from a rabbit before immunization. This is called pre-immune or nonimmune serum. 2. Inject the antigen into the rabbit and wait a few weeks. Inject again and wait. 3. Test the rabbit blood for antibodies against the antigen. Repeat injection if necessary. OVERALL EXPERIMENTAL DESIGN - Grow satellite cells in both growth media and differentiation media - in growth media MEF2 is bound to HDAC - in DM MEF2 is bound to HATs (histone acetyltransferases) - isolate the chromatin/cross-link/sonicate - take some of this for input control. Take the rest and immunoprecipitate with acetylated H4-specific antibody or non-immune serum (serum taken from the same rabbit before antigen treatment, or from a different rabbit) - for all samples, reverse cross-link DNA and PCR amplify with primers to either MCK or myogeninin promoters HYPOTHESIS - acetylation of histones will occur on genes regulated by MEF2 during differentiation in C2 myoblasts - Chromatin around MEF2 sites in MCK or myogenin promoters is closed (unacetylated) in growing C2 myoblasts and open during differentiation of myoblasts into muscle. RESULTS **FIGURE 2A Found that: Acetylated histone H4 is associated with MEF2-responsive regulatory elements during myogenesis Therefore, transcriptional repression is coupled with histone deacetylation This data is consistent with the model that chromatin around MEF2 sites in MCK and myogenin promters is closed (unacetylated) in growing C2 myoblasts and open during differentiation of myoblasts into muscle *** What is wrong with this figure?? LOADING CONTROL NOT EQUAL! CAN HDAC4/5 OVEREXPRESSION INHIBIT MYOGENESIS IN MYOBLASTS? (FIGURE 2B-C) EXPERIMENTAL DESIGN - make stable cell lines over-expressing HDAC: use mouse C2 myoblasts and human HDAC cDNA - mouse vs human HDAC allows to detect exogenous vs endogenous - RT-PCR was performed on the selected genes (on the left of the blots) RESULTS ** FIGURE 2B RT-PCR OVEREXPRESS HDAC4 AND HDAC5 IN C2 MYOBLAST CELL LINES Found: - Late differentiation markers, Muscle Creatine Kinase and Myosin Heavy Chain (data not shown), missing - Early markers, myogenin and -skeletal actin, down-regulated - MEF2C, a late differentiation marker, was lost - MyoD still expressed - Endogenous HDAC4 and 5 expression is constant Therefore, HDAC4 and 5 overexpression inhibits myogenesis What is missing from the figure? - HDAC4 and 5 themselves in the overexpression! This is in the next figure - What is confusing about this figure? 4 How can MEF2C regulate MyoD function before it is expressed? 1) Low levels of MEF2C are present 2) Another MEF2 factor is present and inhibits MyoD function ** - Day 0 = still in GM - note that alpha-skeletal actin arises later on in differentiation - L7 is loading control ** FIGURE 2C SEMI-QUANTITATION OF THE LEVELS OF EXOGENOUS HDAC5 -Used RT-PCR to distinguish between the endogenous (mouse) and exogenous (human) HDAC5 transcripts. - Found: Exogenous levels were 4 times higher than endogenous levels Is this enough of a difference to cause the inhibition of myogenesis?** - Yes, because it produced the effects seen previously - Note exogenous = human HDAC. Endogenous = mouse HDAC - Problems with overexpression of proteins: - High levels of exogenous protein may bind to protein/DNA that the endogenous protein would not bind to. This would lead to an artifact and not a true representation of the endogenous protein function. CAN HDAC INHIBIT MyoD AND/OR MEF2 ACTIVITY IN A
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