The overall goal of this experiment is to exhaustively characterize MyoD protein partners. This method can answer key questions in the skeletal muscle biology field such as molecular regulation of skeletal muscle tonal deformation by the key muscle transcription factor, MyoD. The main advantage of this technique is that it allows us to unravel MyoD partners that have electively low abundance, one localized in one specific subnuclear compartment.
Though this method can provide insight into regulation of the muscle differentiation, it can also be applied to other systems, such as any other tissues, specifically nuclear differentiation. Demonstrating the procedure will be Dr.Lauriane Fritsch, a research assistant from our laboratory. In this protocol, MyoD complexes will be purified from HeLa S3 cells that stably express MyoD with FLAG glutenin tags.
HeLa S3 cells transduced with the empty vector will be used as the control. Both cell lines were previously grown, collected and stored at negative 80 degrees Celsius, as described in the protocol text. Quickly, thaw the cell pellets in a water bath at 37 degrees Celsius.
All buffers and homogenizers should be prechilled and this entire procedure should be performed in a cold room. Typically, 20 grams of cells are used for each experimental point, but for the purposes of this demonstration, only 10 grams, or about 10 mL of cells will be processed. Resuspend the cells in 10 mL of hypotonic buffer A, by using 5 mL of the buffer at first.
Then, adding 2.5 mL, followed by another 2.5 mL. Wash the pipette well with fresh buffer to get the maximum of cells. Transfer 20 mL of the cell suspension into the prechilled homogenizer with a tight fitting pestle.
Homogenize the cells with 20 strokes. Transfer the cell suspension into a 50 milliliter tube. For maximal recovery of cells, wash the homogenizer with 3.5 mL of sucrose buffer.
Transfer this suspension quickly into the 50 mL tube to preserve the nuclei and to limit leakage. Stain a 30 microliter aliquot of the cell suspension with 30 microliters of 4%Trypan Blue and analyze under the microscope to determine the lysis efficiency. All nuclei should be blue if the lysis is successful.
If necessary, repeat the homogenization. Centrifuge for seven minutes at 10, 000 x g and four degrees Celsius to pellet the nuclei. Save the supernatant as a cytoplasmic fraction.
Begin the procedure for preparing the nuclear salt extractable fraction by resuspending the nuclei pellet in 4 mL of sucrose buffer. Then, add 4 mL of high salt buffer drop by drop while mixing thoroughly on a vortex to get a final concentration of 300 mM sodium chloride. Incubate for 30 minutes on ice with mixing every five minutes.
Next, decrease the sodium chloride concentration to 150 mM by adding 8 mL of sucrose buffer. Centrifuge for 10 minutes at 13, 000 x g and four degrees Celsius. Collect the supernatant which is the salt extractable fraction and leave it on ice.
To prepare the chromatin bound fraction, resuspend the pellet meticulously in 3.5 mL of sucrose buffer. Add calcium chloride to a final concentration of 1 mM and mix. The calcium chloride will activate the micrococcal nuclease that will be added later.
Preheat the suspension for one minute at 37 degrees Celsius. Add 35 microliters of a 5 units per microliter micrococcal nuclease stock to get a final concentration of 25 10 thousandth unit per microliter. Incubate for exactly 12 minutes at 37 degrees Celsius.
Mix every four minutes. After 12 minutes, immediately place the reaction on ice. To stop micrococcal nuclease activity, add 56 microliters of 5 molar EDTA pH 8.0 to a final concentration of 4 mM and vortex.
Incubate for five minutes on ice. Sonicate five times for one minute each time at high amplitude allowing a break of one minute between sonications. Alter centrifuge both the salt extractable and the nucleosome enriched fractions for 30 minutes at 85, 000 x g at four degrees Celsius.
Collect the supernatants. The salt extractable fraction should be about 13 mL. And the nucleasome enriched fraction should be about 6 mL.
MyoD protein complexes will be purified by double affinity purification. Since the procedures for FLAG based and HA based purification are similar, only the FLAG based purification will be demonstrated. After adding TEGN transfer the appropriate volume of FLAG resin into a 50 mL tube.
300 microliters of FLAG resin from the commercial 50%stock prewash FLAG resin is needed for each experimental point. Centrifuge for two minutes at 1, 000 x g. Remove the supernatant, add cold TEGN, and centrifuge again.
Wash the FLAG resin with cold TEGN a total of five times. After the last wash, resuspend the FLAG resin in an equal volume of TEGN. For each experimental point, mix 300 microliters of washed FLAG resin and the corresponding protein extract.
Use a 15 mL tube for the nucleasome enriched fraction and a 50 mL tube for the salt extractable fraction. Incubate the tubes on a rotating wheel overnight at four degrees Celsius. On the following day, centrifuge all tubes for two minutes at 1, 000 x g at four degrees Celsius.
Collect the supernatants and keep at four degrees Celsius until the result of the purification efficiency test is obtained. For the salt extractable samples, resuspend the FLAG resin in 4 mL of TEGN and transfer to a 15 mL tube. Repeat this step three times, each time transferring to the same 15 mL to avoid losing beads.
Centrifuge for two minutes at 1, 000 x g at four degrees Celsius. After removing the supernatant, add 13 mL of TEGN to the FLAG resin and centrifuge for two minutes at 1, 000 x g at four degrees Celsius. Wash the resin seven times in this manner.
After the last wash, resuspend the resin in 1 mL of TEGN and transfer into a 1.5 mL low binding tube. Centrifuge for two minutes and remove the supernatant. Resuspend the resin for each experimental point in 100 microliters of FLAG peptide solution at 4 mg/mL at pH 7.8 Mix well and add 100 microliters of TEGN buffer.
Incubate at four degrees Celsius for at least four hours to elute bound proteins. Centrifuge for two minutes and transfer the resin and supernatant to an empty spin column placed in a 2 mL tube. Centrifuge the column for one minute at 6, 000 x g.
Collect the leftover supernatant in the 2 mL tube. Purification efficiency is then tested as described in the protocol text, before proceeding with HA based purification. Shown here are representative results of double affinity purified MyoD complexes from nuclear salt extractable or nucleasome enriched fractions of a HeLa S3 cell line stably expressing FLAG HA MyoD.
Silver staining identified FLAG HA MyoD indicated by the arrow which is absent from the mock cell line. The double affinity purified MyoD complexes were fractionated on glycerol gradients. Western blotting of the fractions using anti-FLAG antibodies uncovered the MyoD subcomplexes in the two subnuclear compartments.
In particular, salt extractable MyoD is distributed in three subcomplexes while the chromatin-bound MyoD belongs mainly to one complex. Mass spectrometry analysis of MyoD interactors isolated from nuclear salt extractable and nucleosome enriched fractions identified a series of known partners and new partners of MyoD. Here the MyoD interactors found in both fractions or, specifically for one of the fractions, are grouped based on their functional annotations.
Some of the mass spectrometry revealed interactors were confirmed by Western blot on MyoD complexes. These include the transcription factors CBF, the SWI/SNF subunit BAF47 and HP1 proteins. Since HeLa cells are not muscle cells and do not normally express MyoD, it is necessary to confirm interactions between newly identified interactors and MyoD and myoblasts.
Nuclear total extracts from proliferating and differentiating mouse myoblasts were used for immunoprecipitation and Western analysis with the indicated antibodies. Once mastered, this technique can be done in two and a half days if it is performed properly. While attempting this procedure, it is important to remember to refine obtained results in myoblasts.
Following this procedure, other methods like knockdown of the particular partner in relevance system like myoblasts, can be performed in order to answer additional questions like functional meaning of the interaction. After it's development, this technique paves the way for researchers in the field of developmental biological epigenetics to explore the regulation network of five transcription factors in most body systems. After watching this video, you should have a good understanding of how to identify the partners of for transcription factor by performing cell fractionation followed by tandem affinity purification coupled with mass spectrometry.
