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Published as doi: 10.1096/fj.07-095000.
 (The FASEB Journal. 2008;22:1694-1701.)
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In vivo interactions of MyoD, Id1, and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer

Jody M. Lingbeck*, Julie S. Trausch-Azar*, Aaron Ciechanover{dagger} and Alan L. Schwartz*,1

* Edward Mallinckrodt Department of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri, USA; and

{dagger} Department of Biochemistry and Rappaport Institute for Research in Medical Sciences, Faculty of Medicine, Technion-Israel Institute for Technology, Haifa, Israel

1Correspondence: Department of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., C.B. 8116, St. Louis, MO 63110, USA. E-mail: schwartz{at}kids.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MyoD, a skeletal muscle transcription factor, is rapidly degraded by the ubiquitin-proteasome system. MyoD interacts with ubiquitously expressed E2A or inhibitor of DNA binding (Id) proteins to activate or inhibit transcription, respectively. Furthermore, MyoD has been shown to modulate the ubiquitin-mediated degradation of Id1 and E2A proteins, E12 and E47. The molecular mechanisms governing these events are not clear but are hypothesized to occur via heterodimer formation. Fluorescence resonance energy transfer (FRET) is a technique for evaluation of protein-protein interactions in vivo. Using acceptor photobleaching FRET and chimeric proteins composed of MyoD, Id1, E12, E47, E12NLS, or MyoDNLS and either cyan fluorescent protein or yellow fluorescent protein, we show that each of the wild-type proteins is capable of homodimerization. In addition, heterodimers form between Id1 and E2A proteins, as well as between MyoD and E2A proteins. The Id1:E2A interaction is stronger than the MyoD:E2A interaction, which is consistent with the notion that inhibition of MyoD action occurs by the sequestration of E2A proteins by Id. The stronger interaction of Id1 with E2A may also explain the decrease in the rate of ubiquitin-proteasome degradation of Id1 that is significantly greater than that of MyoD when E2A proteins are abundant. Thus, these studies extend our understanding of the molecular mechanisms of MyoD action.—Lingbeck, J. M., Trausch-Azar, J. S., Ciechanover, A., Schwartz, A. L. In vivo interactions of MyoD, Id1 and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer.


Key Words: ubiquitin-proteasome degradation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MYOD IS A SKELETAL MUSCLE transcription factor that interacts with Id1 and E2A proteins to inhibit or activate transcription, respectively. We have shown that MyoD when cotransfected with inhibitor of DNA binding (Id) 1 in HeLa cells affects the subcellular localization and decreases the ubiquitin-mediated degradation rate of Id1 (1) . In addition, the E2A proteins, E12 and E47, significantly stabilize MyoD and Id1 from ubiquitin proteasome degradation. Furthermore, MyoD and Id1 increase the stability of the E2A proteins (2) . The increased stability of these proteins may result from heterodimer formation and a reduced ability to serve as a degradation substrate.

Dimerization between MyoD, Id1, and E2A proteins has been reported. The presence of E2A and MyoD homodimers has been detected using various in vitro techniques and in vivo methods for downstream readout (3 4 5 6 7) ; however, reports vary as to the effect of DNA on homodimerization, the strength of the interaction, and the ability of these homodimers to bind to DNA. Homodimers of Id1 have not been detected (8) .

Although homodimers of MyoD and of E2A proteins appear to exist in vitro and in vivo, many reports suggest that heterodimerization is the preferential interaction (3 4 5 , 7 , 9 10 11) and that the heterodimerization between Id1 and E2A proteins is a stronger interaction than that between MyoD and E2A (8 , 9 , 12 , 13) .

Because in vitro studies may not accurately represent what occurs within the cell and the in vivo results reported to date have indicated only subsequent downstream events and not direct protein interaction, we used fluorescence resonance energy transfer (FRET) to determine protein-protein interactions within the cell. FRET involves nonradiative energy transfer within 10–100Å from an excited donor fluorophore to an acceptor fluorophore. Fluorophores must have overlapping spectra to allow for efficient energy transfer from donor to acceptor. Cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), variants of green fluorescent protein (GFP) from Aequorea victoria, is a viable donor acceptor pair for FRET studies.

FRET results in sensitized emission from the acceptor and quenching of the donor emission. Measurement of the acceptor emission is difficult and requires complex corrections because of spectral bleed through and inadvertent excitation of YFP during CFP excitation. An alternative approach is to determine donor dequenching on acceptor photobleaching (acceptor photobleaching FRET). In this case FRET is observed as an increase in donor emission on elimination (bleaching) of the acceptor. Acceptor photobleaching FRET eliminates the need for complex corrections because only donor emission is being determined and the increase in donor signal is not the result of acceptor bleed-through as the acceptor signal is removed during the bleaching.

We have prepared CFP- and YFP-labeled MyoD, Id1, E12, and E47 and their nuclear localization signal (NLS) mutants and demonstrate that heterodimers and homodimers form in both the cytoplasm and in the cell nucleus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Wild-type MyoD and MyoDNLS in the pCIneo vector, Id1 in pcDNA3, E12 in pGK9, and E47 in pCMV have been described previously (2 , 14) . NLS mutants of E12 and E47 have been described elsewhere (2) . pECFP-N1 and pEYFP-N1 were from Clontech Laboratories Inc. (Palo Alto, CA, USA). Dithiobis(succinimidyl)propionate (DSP) used in the cross-linking studies was obtained from Pierce Biotechnology (Rockford, IL, USA).

Cell culture
HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin) (Life Technologies Inc., Gaithersburg, MD, USA) and maintained in a humidified chamber at 37°C and 5% CO2. Transient transfections (efficiency 40–60%) were performed using the FuGENE 6 reagent (Roche Diagnostics Corp., Indianapolis, IN, USA), and cells were analyzed 16–24 h later. Transfection of the CFP- and YFP-labeled constructs into HeLa cells was done so that protein interactions can be seen directly because transfection into muscle cells where they are endogenously expressed may compromise the system by overexpression of these factors.

Myogenic conversion of 3T3-L1 cells
3T3-L1 cells were grown in DMEM supplemented with 10% bovine serum and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin). At 80% confluence, cells were transfected with pCIneo, pCI-MyoD (wild-type), and pCI-MyoD-CFP using PolyFect (Qiagen, Valencia, CA, USA). Cells were grown to 1-day postconfluence, at which time some were changed to "differentiation medium" (DMEM, 10% fetal bovine serum, 10 µg/ml insulin, and 5 µg/ml transferrin). The medium was replaced daily for 7 days in either treatment. Cells were then harvested, and lysates were run on SDS-PAGE and analyzed via Western blot with antimyosin heavy chain (H-300; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by horseradish peroxidase (HRP) -conjugated secondary (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and ECL detection (Pierce Biotechnology).

FRET positive control
A vector containing both CFP and YFP separated by 11 amino acids was prepared by inserting CFP into the pEYFP-N1 vector. More specifically, site-directed mutagenesis was used to mutate the stop codon of CFP in the pECFP-N1 vector and also to introduce a BamH1 site 3' of the CFP coding region. Restriction digest with BamH1 was used to excise the CFP DNA from the vector using the newly mutated BamH1 site and a preexisting BamH1 site in the multiple cloning site (MCS) of the pECFP-N1 vector. The pEYFP-N1 was cut in the MCS with BglII and BamH1, and the excised CFP DNA was then inserted.

Immunolocalization of CFP and YFP constructs
Approximately 16 h after transfection, HeLa cells were fixed in 4% paraformaldehyde and quenched with 0.1 M ethanolamine. Cells were observed with a Zeiss Axioscope microscope, and 10–20 random fields of each culture condition were photographed (x40) using a Zeiss Axiocam digital camera (Carl Zeiss GmbH, Jena, Germany).

Determination of degradation rates in vivo
Approximately 16 h after transfection, HeLa cells were incubated with cycloheximide (CHX) (100 µg/ml; Sigma-Aldrich Corp., St. Louis, MO, USA) to inhibit further protein synthesis. MG132 (20 µM) was added along with CHX as necessary. After incubation, cells were lysed, sonicated, and then centrifuged as described above. The lysates were mixed with an equal amount of 2x Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA), and equal amounts of each sample were run on a 12% Tris-HCl gel (Bio-Rad Laboratories) and were electroblotted onto nitrocellulose (GE Osmonics, Inc., Minnetonka, MN, USA). The blots were probed with rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc.) to MyoD, Id1, E12, or E47, all at 1:100 dilutions or with a rabbit polyclonal antibody to GFP (1:100, BD Biosciences, San Jose, CA, USA). Incubation with a secondary HRP-conjugated antibody followed and detection was by chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The resulting bands were quantitated using the Kodak EDAS system, and the data were graphed using the Excel graphing program (Microsoft Corp, Redmond, WA, USA). Because ubiquitin-mediated degradation is a stochastic process, data were plotted as log of percent pixels vs. time for ease of calculation of the degradation rate. The degradation rate is expressed as half-life (t1/2), the time for degradation of 50% of the sample.

FRET
Approximately 16 h after transfection, HeLa cells were fixed in 4% paraformaldehyde and mounted on sides using the ProLong Antifade mounting medium (Molecular Probes Inc., Eugene, OR, USA), which did not contain the antifade component. A Zeiss LSM510 META confocal microscope (Carl Zeiss GmbH) with a Plan-Apochromat x63/1.4 oil immersion differential interference contrast objective was used for the FRET procedure. The 30.0 mW argon laser was tuned to 458 nm to excite CFP and to 514 nm to excite YFP and an HFT 458/514 dichroic beam splitter was used to deflect the light to the samples. A NFT 515 beam splitter was used to separate the CFP and YFP emission. For image acquisition, a 475–525 band-pass filter was used in the CFP detection channel and a 530 long-pass filter was used in the YFP detection channel.

The acceptor photobleaching method was performed following the method of Gu et al. (15) . Using the bleach module we bleached the YFP signal in defined regions of interest (ROIs) with 514 nm light at 100% power for 200 iterations. The META detector tuned from 467–624 nm was used to measure the intensity in each ROI on 458 nm excitation 5x before and 5x after the bleach. Linear spectral unmixing was used to separate the CFP and the YFP signals. The background was determined by outlining a ROI in a region containing no cells, and ROIs within cells were background corrected before calculation of FRET efficiencies. FRET efficiency (FRETEff) was calculated as (Ipost–Ipre)/Ipost x 100, where Ipre is the intensity of the CFP before the bleach and Ipost is the intensity of the CFP after the bleach. Similar calculations were made for the nonbleached regions as described (16) . The intensities of at least 30 ROIs from at least three different transfections were measured for each protein pair.

Chemical cross-linking
Approximately 16 h after transfection, HeLa cells were washed 2x with cold PBS. DSP (Pierce Biotechnology) was dissolved in dimethyl sulfoxide and added at a final concentration of 500 µM in 2 ml of PBS. Cells were incubated at 4°C for 30 min and washed 2x for 5 min in Tris-buffered saline to block the free amine groups. Cells were then lysed in 200 µl of 1% Triton X-100 in PBS, scraped into an Eppendorf tube (Brinkmann Instruments, Westbury, NY, USA), and incubated on ice for 15 min followed by centrifugation at 14,000 rpm for 10 min at 4°C in an Eppendorf microcentrifuge. Urea was added to a final concentration of 2.5 M, and the samples were heated to 37°C for 10 min. Thereafter, an equal volume of 2x Laemmli sample buffer with and without β-mercaptoethanol (Sigma-Aldrich Corp.) was added to each sample and run on a 12% Tris-HCl gel (Bio-Rad Laboratories) followed by transfer to nitrocellulose (GE Osmonics, Inc.). Blots were probed with a rabbit polyclonal antibody to Id1 (C-20; Santa Cruz Biotechnology, Inc.), MyoD (M-318; Santa Cruz Biotechnology, Inc.), or E47 (N-649; Santa Cruz Biotechnology, Inc.) at a 1:200 dilution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MyoD, Id1, and the E2A proteins are degraded by the ubiquitin-proteasome system (1 , 14 , 17) . For MyoD and Id1, ubiquitination preferentially occurs on the N terminus before degradation by the 26S proteasome (1 , 18) . In addition, rates of degradation vary with subcellular localization (cytoplasm vs. nucleus), at least for MyoD (14) . Thus, to examine in vivo interactions in the cytoplasm and in the nucleus and not alter the degradation pathway MyoD, Id1, and E2A proteins were labeled with a GFP variant protein (CFP or YFP) at the C terminus. As seen in Fig. 1 , the CFP- and YFP-labeled MyoD, Id1, E12, E47, E12NLS, and MyoDNLS localize within the cell identically to that previously seen for the wild-type proteins (1 , 2 , 14) with MyoD, E12, and E47 localizing to the nucleus, Id1 localizing to the nucleus and cytoplasm, and E12NLS and MyoDNLS localizing to the cytoplasm. Furthermore, the rates of degradation of the CFP- and YFP-labeled proteins were similar to those of their wild-type counterparts (data not shown).


Figure 1
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  		Figure 1. Localization of CFP- and YFP-labeled constructs. MyoD, Id1, E12, and E47 were labeled with CFP and/or YFP on the C terminus, transfected into HeLa cells, and visualized.

MyoD, when overexpressed in 3T3-L1 cells, has been shown to induce myogenic conversion into muscle cells as determined by the appearance of myosin heavy chain (19) . To determine whether the labeled proteins were transcriptionally active, MyoD and MyoD CFP were transfected into 3T3-L1 cells (Fig. 2 A), allowed to differentiate and observed for myosin heavy chain. As seen in Fig. 2B , MyoD CFP, like its wild-type counterpart, was able to induce myosin heavy chain expression in these cells and thus is transcriptionally active. Thus, the addition of the CFP moiety to the C terminus of MyoD did not alter its biological activity.


Figure 2
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  		Figure 2. Fluorescently labeled MyoD constructs induce expression of myosin heavy chain in 3T3-L1 cells. 3T3-L1 cells were transfected with wild-type MyoD and with MyoD C-terminally tagged with CFP. A) Lysates from the transfected cells probed for MyoD. B) Lysates from the transfected cells probed for myosin heavy chain.

As a positive control we prepared a construct containing CFP and YFP in the same vector separated by 11 amino acids. Similar controls in which CFP and YFP were separated by 15, 24, and 37 amino acids have been used previously with the highest amount of FRET seen with the shortest linker between the two fluorophores (20) . As seen in Fig. 3 , we also detect a high degree of FRET efficiency with our positive control. FRET efficiency was determined by transfection of a positive control into HeLa cells. Approximately 16 h later the cells were fixed and photobleaching FRET was performed: the nucleus of one cell was bleached with 514-nm light as seen in Fig. 3A (prebleach) and Fig. 3B (postbleach). Using a 458-nm excitation wavelength, the intensity of the pixels in the nucleus was measured 5x before bleaching and 5x after bleaching of the YFP (Fig. 3C ). The resultant spectrum was separated into CFP and YFP channels using spectral unmixing. The increase in CFP emission after photobleaching of the YFP indicates FRET.


Figure 3
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  		Figure 3. FRET-positive control. A positive control for the photobleaching FRET experiments was made by placing CFP into the YFP vector separated by 11 amino acids. The control was transfected into HeLa cells and fixed and photobleaching FRET was performed. A) CFP and YFP emissions before photobleaching on excitation with 458 and 514 nm, respectively. B) Same cells after photobleaching the YFP in the cell nucleus circled. Measurements of the intensity of the spectrally unmixed emissions of CFP and YFP in bleached nucleus were measured 5x before bleaching and 5x after the bleach and plotted in C.

Similar experiments were performed with our CFP- and YFP-labeled proteins. Previously we have shown that E12 directs the cellular localization of Id1 in that Id1 increases from 72 to 99% nuclear when cotransfected into HeLa cells with E12. When the NLS of E12 is mutated (E12NLS), Id1 became only 48% nuclear (2) . In Fig. 4 , E12 or E12NLS, each labeled with CFP, was cotransfected with YFP-labeled Id1 (Fig. 4A, B , respectively). The CFP emission was measured before and after the bleach as described for the positive control. The increase in CFP emission detected in the cell nucleus as seen in Fig. 4C indicates that E12 and Id1 interact within the cell nucleus. Using the NLS mutant of E12 with Id1, we show that these two proteins also interact in the cytoplasm as seen by the increase in CFP emission detected in the cytoplasm after YFP bleaching (Fig. 4D ).


Figure 4
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  		Figure 4. FRET measurements were determined in the cytoplasm and the nucleus. CFP-labeled E12 or E12NLS was cotransfected into HeLa cells with YFP-labeled Id1. Photobleaching FRET was performed as described in the legend to Fig. 3 . A) CFP and YFP emissions of E12 CFP and Id1YFP before and after photobleaching. The bleached nucleus is circled. B) Same emissions for E12NLS and Id1 YFP. C, D) Spectrally unmixed CFP and YFP emissions of E12 CFP with Id1 YFP and E12NLS CFP with Id1 YFP, respectively.

Thus, using this approach and the various chimeric constructs, we examined FRET between pairs of molecules including MyoD:MyoD, Id1:Id1, E12:E12, E47:E47, MyoD:Id1, MyoD:E12, MyoD:E47, Id1:E12, and Id1:E47. We also determined FRET between E12NLS:Id1 and E12NLS:MyoDNLS. FRET efficiencies were calculated for all protein pairs as described in the Materials and Methods section. Pseudoefficiencies were calculated for the nonbleached regions, and the results are summarized in Fig. 5 . The FRETEff values are calculated for the positive and negative FRET controls for both the bleached (Fig. 5A ) and the nonbleached regions (Fig. 5E ). The positive control shows a FRETEff of ~55%. The CFP-only experiments in which no YFP was added show a slight increase in CFP emission after YFP photobleaching (5–8%). There is no obvious explanation for this increase, but this phenomenon has been reported previously (16) . There is also a small amount of FRET seen between CFP-labeled proteins and free YFP. This may be attributable to dimerization of CFP and YFP resulting from high protein concentration after overexpression (21) .


Figure 5
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  		Figure 5. FRET efficiencies. FRET efficiencies were calculated by the formula FRETEff = (Ipre–Ipost)/Ipost x 100%, where Ipre is the intensity before the bleach and Ipost is the intensity after the bleach. A) Graph shows the FRETEff of the bleached regions of the positive and negative controls used in the FRET experiments. Values for E12NLS CFP alone and MyoDNLS CFP alone were determined to be 7.7 ± 0.4 and 9.9 ± 0.5, respectively (data not shown). B, C) FRETEff of the bleached regions of the homodimers and heterodimers, respectively. D) FRETEff calculated for various NLS mutants. E–H) Pseudo-FRETEff values calculated for nonbleached regions. Error bars are reported as ±SEM.

Plotted in Fig. 5B are the calculated FRET efficiencies for MyoD, Id1, E12, and E47 homodimers. The corresponding nonbleached regions are shown in Fig. 5F . Seen here, all of these proteins homodimerized efficiently, including Id1, which was previously thought not to homodimerize (8) . Heterodimer formation is shown in Fig. 5C and the corresponding nonbleached regions in Fig. 5G . Id1 appears to bind more strongly (as seen by the larger FRETEff) to the E2A proteins than to MyoD. All of the determinations in Fig. 5A-G are in the nucleus.

To examine dimerization within the cytoplasm, we prepared NLS mutants of E12 and MyoD. E12NLS and Id1 dimerize efficiently within the cytoplasm (Fig. 5D ). However, dimerization of cytoplasmic E12 and MyoD was not significant (Fig. 5D ). These observations are in agreement with previous observations demonstrating that E12 was able to direct the cellular localization of Id1 but not that of MyoD (2) .

To independently assess homodimer formation of Id1 we used chemical covalent cross-linking (Fig. 6 ). HeLa cells were transfected with Id1 and treated with or without the amine cross-linker DSP. Treatment with DSP produces a band at 32 kDa, the predicted size of a homodimer of Id1. Treatment of the cross-linked product with β-mercaptoethanol cleaved the linker arm and reduced the band to 16 kDa, corresponding to a monomer of Id1.


Figure 6
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  		Figure 6. Id1 cross-linking. Id1 was transfected into HeLa cells and ~16 h after transfection the cells were treated with or without DSP to cross-link the protein for 30 min. The cells were lysed and half run on a SDS-PAGE gel with and without β-mercaptoethanol (BME) treatment.

To independently assess heterodimer formation of MyoD and Id1 we extended the chemical covalent cross-linking approach. HeLa cells were transfected with MyoD and Id1 and treated with or without DSP. Potential dimers include MyoD:Id1, MyoD:MyoD, and Id1:Id1. As seen in Fig. 7 , treatment with DSP produces a band at ~140 kDa seen with both anti-MyoD and anti-Id1. Treatment of the cross-linked product with β-mercaptoethanol reduced the band to its MyoD (46 kDa) and Id1 (16 kDa) monomer components (Fig. 7) . The size of the cross-linked product (~140 kDa) may represent either a complex of MyoD:Id1 dimers or a complex of MyoD:Id1 plus additional ancillary proteins not identified by the antibodies to MyoD or Id1. Resolution of this complex will await future studies. A faint band at ~32 kDa is present in Fig. 7A , probably representing Id1 homodimers. An even less prominent band at ~92 kDa representing MyoD homodimers is seen in Fig. 7B .


Figure 7
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  		Figure 7. MyoD-Id1 cross-linking. MyoD and Id1 were transfected into HeLa cells and ~16 h after transfection the cells were treated with or without DSP to cross-link the protein for 30 min. The cells were lysed and half were treated with and without β-mercaptoethanol (ME) (noted in figure). Aliquots of each sample were run on Western blots probed with anti-Id1 (A) or anti-MyoD (B). The monomers of Id1 and MyoD are noted with open arrows on the respective blots. The complex that contains both MyoD and Id1 is noted by a closed arrow in both panels. Homodimers of Id1 and MyoD are noted by an open arrowhead on each respective panel.

Assessment of E47:Id1 heterodimer formation was also performed with chemical covalent cross-linking in a manner analogous to that described above for MyoD and Id1. As seen in Fig. 8 , treatment with DSP produces a wide band at ~120–145 kDa seen with both anti-E47 (72 kDa) and Id1 (16 kDa) monomer components (Fig. 8) . The size of the cross-linked product (~120–145 kDa) may represent a complex of E47:Id1 plus additional ancillary proteins not identified by the antibodies to E47 or Id1.


Figure 8
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  		Figure 8. E47-Id1 cross-linking. E47 and Id1 were transfected into HeLa cells, and ~16 h after transfection the cells were treated with or without DSP to cross-link the protein for 30 min. The cells were lysed and half were treated with and without β-mercaptoethanol (ME) (noted in figure). Aliquots of each sample were run on Western blots probed with anti Id1 (A) or anti-E47 (B). The monomers of Id1 and E47 are noted with open arrows on the respective blots. The complex that contains both E47 and Id1 is noted by a closed arrow in both panels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Transcription factors, many oncoproteins, and a variety of other regulatory proteins are synthesized in the cytoplasm and translocated into the nucleus where they exert biological effects. Much of the action of these proteins is thought to be enhanced or inhibited via heterodimer formation with other nuclear proteins. For example, MyoD, a skeletal muscle transcription factor is inhibited by Id1 and activated by association with E2A proteins, E12 and E47. Determination of dimer formation has generally relied on in vitro assays, which may or may not mimic actual events within the cell, or by detecting downstream events in in vivo assays.

For example, using coimmunoprecipitation assays Benezra et al. (9) also demonstrated in vitro that Id binds more strongly to the E2A proteins than to MyoD. Using a yeast two-hybrid assay along with coimmunoprecipitation assays Langlands et al. (13) demonstrated that Id interacted more strongly with E2A proteins than with MyoD or other myogenic factors. They were also able to demonstrate in vivo that Id proteins disrupted E2A:MyoD complexes as determined by the reduced ability of E2A:MyoD to transactivate a MCK-CAT reporter in the presence of Id. Paulmurugan et al. (22) reported an interaction of MyoD and Id in cell culture and in mice using a split firefly luciferase assay. These assays, however, have been unable to define how these proteins interact and where in the cell the interaction occurs.

FRET measures the energy transfer between a donor and acceptor and can be used to determine the location and distance between directly interacting proteins when the proteins are labeled with an appropriate donor acceptor pair such as CFP and YFP. However, FRET is hampered by the need for complex corrections for spectral overlap, donor or acceptor bleed-through, and inadvertent excitation of YFP on excitation of CFP. To alleviate many of these issues acceptor photobleaching FRET may be used. This process measures the donor emission before and after bleaching of the acceptor. The emission of the donor increases after bleaching of the acceptor because energy is no longer being transferred to the acceptor molecule. Acceptor photobleaching FRET is also hampered from issues with spectral bleed-through, but these issues may be resolved via spectral unmixing (15) .

As described above, using photobleaching FRET with spectral unmixing, we show that 1) MyoD, Id1, E12, and E47 readily form homodimers in the cell nucleus; 2) heterodimer formation occurs between MyoD and E12 or E47, between Id1 and E12 or E47, and between MyoD and Id1, also in the cell nucleus; and 3) heterodimer formation between Id1 and E12NLS but not between MyoDNLS and E12NLS occurs in the cytoplasm.

These FRET results also demonstrate stronger binding of Id1 to E12 or to E47 than to MyoD and a stronger Id1:E2A interaction compared with the MyoD:E2A interaction (Fig. 5) . These results are consistent with the notion that the inhibitory effects of Id1 are not due to binding to MyoD but rather to the sequestration of E2A proteins, which prohibits them from binding to MyoD and activating transcription. Furthermore, MyoD, which functions in the nucleus, appears to only form active heterodimers with E12 within the nucleus, perhaps facilitated by the presence of DNA. In contrast, Id1 which does not bind DNA, dimerizes with E12 both in the cytoplasm and in the nucleus. Thus, the observation that Id1 and E2A interact both in the cytoplasm and in the nucleus suggests that Id1 and E2A may regulate numerous biological processes in both of these cellular compartments.

The lack of heterodimer formation of the transcriptionally active species (i.e., MyoD:E2A) in the cytoplasm is probably not restricted to myogenic regulatory factors but may also apply to other regulatory transcription factors that are active only in the nucleus. Previous studies have shown that MyoD regulates the ubiquitin proteasome-mediated degradation of Id1 (1) . Our FRET results indicate that heterodimer formation occurs between MyoD and Id1. E2A proteins significantly stabilize the degradation of MyoD and, to a greater extent, Id1 (2) . Consistent with this finding, our FRET results show a stronger interaction between E2A proteins and Id1 compared with that with MyoD. These results taken together suggest that the decreased rates of protein degradation of these factors via the ubiquitin proteasome pathway are probably a result of heterodimer formation. Thus, we show that heterodimer formation of these protein pairs can differ between the cytoplasm and the nucleus, which is likely to be an important factor in their degradation as the functionality of the ubiquitin proteasome system also differs between the cytoplasm compared with the nucleus.


   ACKNOWLEDGMENTS
 
We thank Wandy Beatty and Darcy Gill for assistance with FRET software and Guojun Bu for critical review. This study was supported by the U.S. National Institutes of Health (A.L.S.).

Received for publication August 1, 2007. Accepted for publication December 6, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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