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Shrimati Datta,2,
Lih-Huei L. Liaw,1
Cristen J. Wu,1
Gopi Manthripragada,1,
Timothy F. Osborne,2 and
Vickie J. LaMorte1*
Laser Microbeam and Medical Program, Beckman Laser Institute, Department of Surgery,1 Department of Molecular Biology and Biochemistry, University of California, Irvine, California2
Received 10 September 2004/ Returned for modification 15 October 2004/ Accepted 4 January 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SREBPs are unique in that they remain inactive through sequestration in the membrane of the endoplasmic reticulum by two membrane-spanning domains. Depletion of sterols triggers their translocation to the Golgi, where a two-step proteolytic process releases the amino-terminal half from the membrane anchor, and this mature transcription factor is then translocated to the nucleus, where it activates genes involved in regulating lipid balance (9, 10).
SREBPs, like other bHLH-LZ transcription factors, dimerize through their HLH-LZ motif and bind DNA through their basic domain. The results of overexpression studies with dominant negative versions of the SREBPs have shown that SREBP-1a and -2 can dimerize with each other, although not with the other bHLH-LZ family members tested so far (24). However, the localization, putative formation, and activity of SREBP homo- versus heterodimers has not been directly evaluated or compared in any systematic way. Because both the SREBP-1 and -2 proteins are expressed in most tissues and cell types, the function of the homo- versus heterodimer is an important question. In the present studies, we investigated the occurrence and spatial distribution of exogenously expressed SREBP-1a and -2 homo- and heterodimers in vivo and demonstrated that both homo- and heterodimers are transcriptionally active in the cell. These findings indicate significant differences in the subnuclear distribution of SREBP-1a and -2 and have major implications for gene activation mediated by the SREBP family.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ii) PML-GFP fusion. The GFP coding sequence was generated by PCR with pEGFP-N1 as the template and oligonucleotides containing an MluI site at the 5' end and an XbaI site at the 3' end. The fragment was inserted into the previously described CMX-PML vector (13), which was digested with MluI and NheI at the C terminus.
(iii) SREBP fusion plasmids for fluorescence resonance energy transfer (FRET) analysis. For SREBP-1aCFP and SREBP-2CFP, full-length mature SREBP-1a and -2 were digested with BamHI and HindIII from pPacSREBP-1a and -2, respectively, as described above and cloned into pECFP-N1 (Clontech) digested with BglII and HindIII. For SREBP-1aYFP and SREBP-2YFP, full-length mature SREBP-1a and -2 were digested with EcoRI and HindIII from CMV-SREBP-1a and -2, respectively, as previously described (16) and cloned into EcoRI- and HindIII-digested yellow fluorescent protein (YFP; Topaz; Packard BioSciences, Meriden, Conn.).
(iv) Tethered-dimer fusions. To construct the SREBP-1a tethered-dimer fusion (designated SREBP-1a/SREBP-1a), first a double-stranded oligonucleotide containing an 18-amino-acid tether was digested with EcoRI and ligated upstream of EcoRI-digested pCMV-5 containing mature SREBP-1a (described above). Next, an Xba fragment containing the tether and SREBP-1a fusion was subcloned downstream of Xba-digested plasmid pCDNA3.1+ (Invitrogen, San Diego, Calif.) containing two copies of the Flag epitope sequence upstream of the SREBP coding sequence. The other tethered SREBP dimer fusions were made similarly. SREBP-2/SREBP-2 represents the SREBP-2 homodimer. SREBP-1a/SREBP-2 and SREBP-2/SREBP-1a represent the SREBP-1a and -2 heterodimer, with SREBP-1a at the amino terminus for the SREBP-1a/SREBP-2 fusion and SREBP-2 at the amino terminus for the SREBP-2/SREBP-1a fusion.
(v) Reporter gene plasmid. The construction of the reporter plasmid for the wild-type low-density lipoprotein receptor (LDL-R)-encoding gene has been described previously (26).
Cell culture, microinjection, and transient-transfection assays. (i) Microinjection. HEp-2 cells (American Type Culture Collection, Manassas, Va.) were cultured as previously described (12) in drilled 35-mm-diameter dishes with an alpha-numerically gridded coverslip (Bellco Glass, Vineland, N.J.) affixed to the bottom. Approximately 10–15 liters of DNA plasmid per cell (200 ng/µl) was introduced into the nucleus of the cell with a semiautomated microinjection system (Eppendorf Scientific, Westbury, N.Y.) and a Zeiss Axiovert microscope (Zeiss Inc., Oberkochen, Germany). For the FRET studies, DNA plasmid concentrations were kept equal for cyan fluorescent protein (CFP) and YFP to ensure equivalent expression within a cell. Cells were incubated overnight for all studies to permit expression. Each experimental condition was performed in triplicate.
(ii) Activation studies. CV-1 cells (from Ken Cho, University of California, Irvine) were grown in Dulbecco's modified Eagle's medium (Irvine Scientific, Irvine, Calif.) with 10% fetal bovine serum and additives purchased from Invitrogen (normal medium). Cells were plated in 6-mm wells at 60,000 per well on day 0. Cells were transfected by calcium phosphate coprecipitation on day 1. On day 2 (12 to 16 h posttransfection), cells were washed three times with 1x phosphate-buffered saline (PBS) and refed with normal medium. Cells were harvested on day 3 (24 h posttransfection) with cell lysis buffer (25 mM Gly-Gly, 15 mM MgSO4, 4 mM EGTA, 0.25% Triton X-100) and analyzed for luciferase and ß-galactosidase activities as described previously (2). Each experimental point was performed in duplicate and represents several independent transfections.
(iii) Dominant negative studies. CV-1 cells were grown as described above. Cells were plated in 60-cm-diameter dishes at 125,000 per dish on day 0. On day 1, cells were transfected by calcium phosphate coprecipitation. On day 2 (16 to 18 h posttransfection), the cells were washed three times with 1x PBS and refed with normal medium. Cells were harvested by a standard freeze-thaw method as described previously (1) on day 3 and analyzed for luciferase and ß-galactosidase activities as described above. The data represent the average of two separate transfection experiments, each performed in duplicate.
(iv) Tethered-dimer activation studies. 293T cells (from Craig Walsh, University of California, Irvine) were grown as described for CV-1 cells above. Cells were plated in six-well dishes at 3.5 x105 per well on day 0. Cells were transfected by calcium phosphate coprecipitation on day 1. On day 2 (12 to 16 h posttransfection), cells were washed two times with 1x PBS and refed with normal medium containing cholesterol and 25-hydroxycholesterol (Toruloides, Inc., Newport, R.I.) at final concentrations of 12 and 1 µg/ml, respectively, to suppress endogenous SREBP activity. Cells were harvested on day 3 (30 h after refeeding) with cell lysis buffer and analyzed for luciferase and ß-galactosidase activities as described above. Each experimental data point was performed in duplicate and represents several independent transfections.
(v) Western blot analysis of protein expression. 293T cells were plated on day 0 in 60-cm-diameter dishes at 6 x 105 per dish and transfected on day 1 by calcium phosphate coprecipitation. On day 2 (12 to 16 h posttransfection), the cells were washed two times with 1x PBS and refed with normal medium. Cells were harvested on day 4 as described previously (31). Extracts were assayed for ß-galactosidase activity as described above. Equivalent amounts of protein, normalized to ß-galactosidase values, were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by immunoblotting. SREBP-1a, GFP, and non-GFP-tagged fusions were detected with an antibody, IgG2A4, to SREBP-1a (sc-13551; Santa Cruz, Santa Cruz, Calif.). SREBP-2, GFP, and non-GFP-tagged fusions were detected with an antibody, IgGID2, to SREBP-2 (American Type Culture Collection). SREBP-tethered-dimer fusions were detected with an antibody to the Flag epitope (Sigma, St. Louis, Mo.).
Antibodies and immunostaining. (i) PML and SUMO-1 immunodetection. HEp-2 cells microinjected with SREBPGFP fusions were fixed in 3.7% formaldehyde-PBS and immunostained with an affinity-purified rabbit antibody (6) (a kind gift from J. Dyck) as previously described (12). Similarly, cells were immunostained with a mouse monoclonal antibody to SUMO-1 (Zymed Laboratories, San Francisco, Calif.).
(ii) GFP immunodetection. HEp-2 cells microinjected with SREBP-2GFP were fixed and immunostained prior to electron microscopy analysis (19) with affinity-purified mouse anti-GFP (Clontech) at a 1:100 dilution. Primary antibodies were subsequently detected with a 1.4-nm Nanogold-conjugated secondary goat antibody and a silver enhancement process (Nanoprobes, Yaphank, N.Y.).
Imaging and microscopy. (i) Low-light fluorescence microscopy imaging. Samples were examined with a Zeiss Axiovert 10 microscope equipped with a 100-W Hg lamp excitation source and a 100x Zeiss Plan-NEOFLUAR objective (numerical aperture = 1.2). A slow-scan cooled charge-coupled device (CCD) camera (576 by 384 pixels, model 57-180; Princeton Instruments, Trenton, N.J.) with a 16-bit-per-pixel dynamic range of data acquisition interfaced with a personal computer was attached to the microscope. GFP fluorescence was excited with a 480- to 30-nm band-pass filter and detected with a 535- to 40-nm band-pass filter. Texas Red fluorescence for PML detection was excited with a 560- to 40-nm band-pass filter and detected with a 630- to 60-nm band-pass filter. Images were acquired with IPLab, version 3.5.5 (Scanalytics, Fairfax, Va.), and converted to TIFF files. TIFF files were subsequently processed with ADOBE Photoshop, version7.0.
(ii) Two-photon imaging and spectroscopy FRET. The two-photon imaging and spectroscopy system used for this study has been described previously (13, 32). Briefly, it consists of a mode-locked Ti:Sapphire laser (170-fs pulse width, 76-MHz repetition rate; Mira 900F; Coherent, Santa Clara, Calif.) pumped by a 5-W Verdi laser (Coherent). The beam exiting the laser is deflected into the back port of an inverted Axiovert 100 microscope (Zeiss Inc.) and scanned across the sample via a personal-computer-controlled, galvanometer-driven x-y scanner (Series 603X; Cambridge Technology, Watertown, Mass.). The beam is reflected by a short-pass 675-nm dichroic beam splitter (Chroma Technology Corp., Brattleboro, Vt.) and focused onto the sample with a 100x oil immersion microscope objective (numerical aperture = 1.45; Zeiss Inc.). The excitation wavelength was 810 nm, which has been demonstrated to minimally excite YFP (13). The average power entering the microscope is approximately 30 mW (2.5 mW at the sample site). Two-photon-excited fluorescence from the sample is epicollected, discriminated with the dichroic beam splitter, filtered by a short-pass 600-nm filter (CVI Laser Corp., Livermore, Calif.), and detected. Two-dimensional (x-y plane) images (256 by 256 pixels) are acquired at a rate of 1 frame/s (pixel dwell time of 16 µs/pixel) with a single-photon counting photomultiplier tube (Hamamatsu Corp., Bridgewater, N.J.) and cover an area of 20 by 20 µm for the 100x microscope objective. An SBG39 wide-pass (322 to 654 nm) blue-green emission filter (CVI, Albuquerque, N.Mex.) is placed in front of the photomultiplier tube. Spectra are obtained with a SpectraPro-150 spectrograph equipped with a 300-groove/mm grating blazed at 500 nm (Acton Research Corp., Acton, Mass.) and a high-dynamic-range MicroMAX:512BFT CCD camera (Princeton Instruments), which is controlled by an ST-133 controller (Princeton Instruments). The spectrograph and camera settings are personal computer controlled through commercially available software (WinSpec/32, version 2.4.6.6; Roper Scientific, Inc., Trenton, N.J.). The CCD temperature is maintained at the minimum possible temperature (–45°C) for all of the experiments to ensure a low dark-noise level. The entrance slit of the spectrograph is set to a width of 0.5 mm. The spectrum acquisition time was 60 s. Switching between imaging and spectrum acquisition is achieved by changing the position of a built-in microscope mirror. When both two-photon images and spectra are acquired from the sample, the two-photon images are acquired and stored and then the emission spectrum is immediately acquired from the same depth (z) in the sample.
Autofluorescence of the cell is negligible. Dark-noise spectra are subtracted from the acquired sample spectra. Acquired images (256 by 256 pixels) were converted to TIFF format with IPLab, version 3.5.5 (Scanalytics), with no postprocessing modifications. Spectral data were imported into Microsoft Excel 2000 (Microsoft Corp.) and plotted as the intensity (arbitrary units) versus the emission wavelength (nanometers). Because of the low signal-to-noise ratio in the acceptor-alone case, a moving average (period = 10) function was used to plot the spectra. Peak intensity ratios were statistically analyzed by separate variance t tests, and a P value was calculated.
Transmission electron microscopy. Ultrastructural analysis of pre-embedded immunostained serial thin sections (60 nm) of single microinjected cells was performed as previously described (15) on a Tecnai 12 BioTwin electron microscope (FEI Company, Hillsboro, Oreg.).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FRET. All previous studies that have evaluated SREBP function have assumed the formation of homodimers when the monomeric expression constructs are individually expressed in cells or animals. However, these studies have all been performed with cells in which the potential for endogenous SREBPs or other bHLH proteins to dimerize with the expressed protein exists. The only evidence for potential heterodimer formation comes from the interpretation of dominant negative overexpression studies. To date, there are no previous studies that have directly evaluated the ability of SREBPs to form homo- or heterodimers inside a living cell.
To examine the occurrence of heterodimers and homodimers between exogenously expressed SREBP-1a and -2 in vivo, we used a spectroscopic approach to monitor FRET at the subcellular level. CFP and YFP variants were used as the FRET donor-acceptor pair. Expression vectors encoding the mature domain of either SREBP-1a or SREBP-2 at the N terminus and either CFP or YFP at the C terminus were microinjected into HEp-2 cells either alone or in combination to monitor the presence of FRET. In general, SREBP-1a and -2 fusion proteins with CFP or YFP were localized to the nucleus and excluded from nucleoli. To evaluate localization and dimerization, two-photon fluorescence images were captured and the corresponding spectra of cells expressing the donor alone (SREBP-1aCFP), the acceptor alone (SREBP-2YFP), and both as homodimers (SREBP-1aCFP/SREBP-1aYFP, SREBP-2CFP/SREBP-2YFP) or as heterodimers (SREBP-1aCFP/SREBP-2YFP) were acquired with an excitation wavelength of 810 nm. Figure 2 shows images and the corresponding spectra from representative cells of each condition.
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In agreement with Fig. 1, SREBP-2 homodimer fluorescence is confined to the nucleus and may either exhibit a diffuse pattern (not shown) or be localized to discrete domains, ranging from a speckled pattern (Fig. 2d) to larger foci (Fig. 2e). The spectra corresponding to the images in Fig. 2d and e display the characteristic donor CFP peaks at 476 and 501 nm and an increased signal at 527 nm compared to the donor spectrum alone. Similar spectra were obtained for cells exhibiting a diffuse fluorescence pattern. When fluorescence exhibits a speckled pattern (Fig. 2d), the ratio of 501 to 476 nm was 0.96 whereas the FRET ratio, 527 to 476 nm, was increased to 1.40, which is indicative of homodimer formation. When fluorescence is localized to larger discrete foci (Fig. 2e), the ratio of 501 to 476 nm is maintained at 0.96 whereas the FRET ratio, 527 to 476 nm, is increased to 1.89.
When examining SREBP-1a/SREBP-2 heterodimer interactions, three distinct patterns of fluorescence are observed. Figure 2f and g exhibit fluorescence patterns similar to that of SREBP-2 homodimer formation, where discrete nuclear domains are present. In the spectrum corresponding to the image of Fig. 2f, the ratios of 501 to 476 and 527 to 476 nm are 0.92 and 1.07, respectively. In the spectrum corresponding to the image of Fig. 2g, the ratios of 501 to 476 and 527 to 476 nm are 0.92 and 1.09, respectively. In contrast, cells exhibiting only a diffuse nuclear fluorescence pattern (Fig. 2h) had a ratio of 527 to 476 nm of 0.56. The ratio of 501 to 476 nm is consistent at 0.92. For each spectrum (Fig. 2c to h), the normalized donor spectrum (Fig. 2a) was subtracted, yielding the net FRET response shown in Fig. 3.
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100GFP, 88% maintained a speckled or focal fluorescence pattern (Fig. 7d), which is similar to what was observed with the wild-type SREBP-2GFP construct (Fig. 1b). However, with SREBP-2150GFP, 92% of the cells (n = 257) displayed a diffuse fluorescence pattern in the nucleus. Similarly, for all cells expressing SREBP-2200GFP (n = 106), SREBP-2250GFP (n = 140), or SREBP-2300GFP (n = 149), fluorescence presented a diffuse nuclear pattern. An internal deletion of amino acids 101 to 249 also resulted in diffuse nuclear fluorescence (data not shown). Truncation of the first 330 amino acids (SREBP-2330GFP) resulted in cells with diffuse fluorescence in both the nucleus and the cytoplasm (n = 174). Taken together, these data indicate that deletion of amino acids 101 to 149 disrupts a domain that is responsible for localizing expressed SREBP-2 into a speckled or focal pattern and that the nuclear localization signal for SREBP-2 is disrupted when amino acids 301 to 330 are removed.
Structural characterization of foci.
An array of nuclear proteins has been shown to localize to speckles or foci within the cell, while fewer have been characterized to localize to discrete nuclear structures or physically identifiable bodies. In this regard, we noted a similarity in appearance between the SREBP-2 foci and the well-characterized PML-containing nuclear body (6, 12, 25). Thus, we wanted to evaluate whether there was a relationship between the two. First, we performed indirect immunofluorescence studies with corresponding antibodies and expression constructs to see if the SREBP-2 and PML foci were identical. In 100% of cells (n = 136) expressing SREBP-1aGFP, immunostaining for endogenous PML resulted in a normal pattern of PML localization corresponding to an average of 10 to 20 PML bodies per nucleus (Fig. 8a and b). In contrast, endogenous PML staining in cells expressing SREBP-2GFP showed a significant loss or depletion of PML staining (96%, n = 276) from the corresponding bodies (Fig. 8c and d). In some cases (4%), PML could be observed tracking out of the PML-containing nuclear bodies (Fig. 8e and f). To further determine whether expression of SREBP-2 results in loss or depletion of PML from its nuclear bodies, we coexpressed untagged SREBP-2 along with PMLGFP. This resulted in a decrease or absence of PML fluorescence within its corresponding bodies (data not shown). Similarly, cells expressing SREBP-2100GFP exhibited a loss or depletion of PML-containing nuclear body fluorescence (93%, n = 118) (Fig. 8g and h). In some instances (7%), colocalization of SREBP-2GFP and PML was observed (data not shown). In contrast, expression of the N-terminal deletion constructs of SREBP-2 that exhibited a diffuse pattern on their own did not alter PML localization (data not shown). In addition, similar studies with SUMO-1 to identify the PML-containing nuclear bodies resulted in the same staining patterns (data not shown). Taken together, these data suggest that SREBP-2 foci may correspond to the well-characterized PML bodies and demonstrate that there is a dynamic relationship between the nuclear body localization of SREBP-2 and components of the PML-containing nuclear body.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bHLH-LZ domains of SREBP-1 and -2 are highly conserved (74% identity), and a recent report has suggested that overexpression of a dominant negative SREBP-1a protein can decrease the activity mediated by both SREBP-1a and -2 (24). However, a direct demonstration that the two proteins actually form heterodimers in cells and the activity mediated by the individual homodimers relative to that mediated by a putative heterodimer has not been directly investigated.
In the present studies, we have used novel imaging techniques including two-photon-excited imaging and spectroscopy FRET coupled with key molecular biology methods in which we connected individual SREBP isoforms together into tethered dimers (18) to analyze the subnuclear spatial localization and function of SREBP homo- and heterodimers.
These data support the in vivo occurrence and uncover distinct subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers and provide potential insight into their differential regulation. By using two-photon imaging and spectroscopy FRET to examine protein-protein interactions in subfemtoliter volumes in vivo (13), we demonstrated that when exogenously expressed SREBP-1a homodimerizes in vivo, it is localized diffusely in the nucleus. FRET signals from similarly expressed SREBP-2 homodimers were detected both within a diffuse fluorescence pattern and in discrete nuclear domains ranging from speckles to larger foci. Importantly, SREBP-1a homodimers never exhibited a speckled or focal pattern in our studies. These techniques also provide novel evidence to directly support the existence of SREBP-1a and -2 heterodimers in vivo. Intense FRET signals from heterodimers were detected within cells exhibiting fluorescence in discrete nuclear domains. Notably, there was a significant FRET signal decrease in cells in which both SREBPs were expressed, and the fluorescence was present in a diffuse nuclear pattern, strongly suggesting that heterodimers are residing inside the discrete nuclear domains.
The FRET analysis data correlate with the different nuclear localization patterns observed for the individual GFP-tagged versions of mature SREBP-1a and -2. SREBP-1aGFP was distributed diffusely throughout the nucleus, whereas SREBP-2GFP expression was localized in subnuclear domains, exhibiting a speckled or focal pattern in the majority of cells. The FRET studies indicated that the SREBP-1a and -2 heterodimers were localized in subnuclear domains similarly to SREBP-2 homodimers, suggesting that SREBP-1a localization was influenced by the coexpression of SREBP-2. When untagged SREBP-2 was coexpressed, it also resulted in the redistribution of SREBP-1aGFP in a speckled or focal pattern. Taken together, these results indicate that SREBP-2 coexpression results in recruitment of SREBP-1aGFP into speckles or foci, indicating that SREBP-2 can affect the localization of SREBP-1a in a dominant fashion.
Our two-photon imaging and spectroscopy FRET studies indicate that both SREBP homo- and heterodimers occur in living cells, but they do not evaluate the functional significance of the homo- versus heterodimer. In order to accurately measure the activation potential of SREBP homo- and heterodimers and avoid complications from endogenous proteins, we used a tethered-dimer strategy. By linking two SREBP monomers with a flexible polypeptide spacer, we could minimize any potential dimerization with endogenous proteins, which is a significant complication when constructs expressing only the monomeric SREBPs are analyzed. We constructed tethered dimers of SREBP-1a and -2 in both orientations and analyzed their abilities to activate the LDL-R promoter in a luciferase reporter-based transactivation assay. The results show for the first time that the SREBP homo- and heterodimers activate transcription to similar levels. Similar results were obtained for other SREBP target promoters (5).
Our studies provide clear and significant new information about differential trafficking of exogenously expressed SREBP isoforms in intact cells. Because the exogenously expressed SREBP-GFP fusion proteins may display different localization patterns than the corresponding endogenously expressed proteins, it will be important to extend these observations to determine if endogenously expressed proteins behave similarly. However, these additional studies await the development of more specific antibodies and modified imaging techniques to evaluate the low levels of endogenously expressed SREBPs.
A recent report suggests that dimerization of SREBPs occurs prior to transport into the nucleus (14). When combined with our data, these results indicate that upon nuclear import, dimers are differentially directed to discrete locations, and more specifically in the case of SREBP-2, to discrete electron-dense nuclear structures. The dynamic interplay between SREBP-2 and PML/SUMO-1 trafficking suggests that they may reside in the same bodies. In agreement with previous findings, PML-containing nuclear bodies may serve as functional complexes whose constituents are dynamic, i.e., CBP (4, 12) and, as shown here, SREBP-2 dimers.
We have also demonstrated that amino acids 101 to 149 are necessary for expressed SREBP-2's ability to localize within speckles or foci. A previous report suggested that SREBP-1a and -2 were recruited to punctate foci only when p300 was also coexpressed (8). p300 interacts with the SREBP-activation domain (30), which is located at the extreme amino terminus of both SREBP-1a and -2. Our studies indicate that SREBP-2 localizes to foci independently of p300 overexpression and is independent of the amino-terminal activation domain as SREBP-2100GFP was efficiently localized to foci. Fluorescence cross-correlation spectroscopy would allow monitoring of the changes in complex diffusion rates and the differences in the molecular sizes of the mobile SREBP-coactivator complexes to definitively determine the participants in these complexes and where these interactions occur.
Overall, our results demonstrate that there are differences in the spatial localization of homodimers and heterodimers between exogenously expressed SREBP-1a and -2. Additionally, our data provide the basis for further studies to understand the mechanism and functional significance of this differential localization. Because the dimerization specificity for the individual bHLH proteins can have a significant impact on function, it will be important to evaluate the dimerization and activation properties of other family members with a methodology similar to the one used in the present study.
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ACKNOWLEDGMENTS |
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This work was supported by the National American Heart Association (993005N to V.L.); the National Institutes of Health (HL48044 to T.O.); the National Institutes of Health Research Resource, Laser Microbeam, and Medical Program (P41R01192); and the Air Force of Scientific Research, Medical Free Electron Laser Program (FA9550-04-1-01-01).
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FOOTNOTES |
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A.Z. and S.D. contributed equally to this work.
Present address: Department of Medicine, Georgetown University, Washington, D.C.
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