Id Proteins Negatively Regulate Basic Helix-Loop-Helix Transcription Factor Function by Disrupting Subnuclear Compartmentalization

Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect. Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect. Id proteins function as global regulators of cell fate determination. They play a pivotal role in the coordinate regulation of gene expression during cell growth/cell cycle control, differentiation, and tumorigenesis (reviewed in Refs. 1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar and 2Benezra R. Oncogene. 2001; 20: 8288-8289Crossref Google Scholar). Recent studies have also highlighted their role in cellular senescence (3Alani R.M. Young A.Z. Shifflet C.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7812-7816Crossref PubMed Scopus (184) Google Scholar, 4Ohtani N. Zebedee Z. Huot T.J.G. Stinson J.A. Sugimoto M. Ohashi Y. Sharrocks A.D. Peters G. Hara E. Nature. 2001; 409: 1067-1070Crossref PubMed Scopus (524) Google Scholar) and in cell fate decisions in cells of specialized lineages such as lymphocytes (5Sugai M. Gonda H. Kusunoki T. Katakai T. Yokota Y. Shimizu A. Nat. Immunol. 2003; 4: 25-30Crossref PubMed Scopus (108) Google Scholar) (reviewed in Ref. 6Engel I. Murre C. Nat. Rev. Immunol. 2001; 1: 193-199Crossref PubMed Scopus (160) Google Scholar), vascular endothelial cells (7Lyden D. Young Z.A. Zagzag D. Yan W. Gerald W. O'Reilly R. Bader B.L. Hynes R.O. Zhaung Y. Manova K. Benezra R. Nature. 1999; 401: 670-677Crossref PubMed Scopus (769) Google Scholar, 8Volpert O.V. Pili R. Sikder H.A. Nelius T. Zaichuk T. Morris C. Shiflett C.B. Devlin M.K. Conant K. Alani R.M. Cancer Cell. 2002; 2: 473-483Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and neuronal cells (7Lyden D. Young Z.A. Zagzag D. Yan W. Gerald W. O'Reilly R. Bader B.L. Hynes R.O. Zhaung Y. Manova K. Benezra R. Nature. 1999; 401: 670-677Crossref PubMed Scopus (769) Google Scholar, 9Martinsen B.J. Bonner-Fraser M. Science. 1998; 281: 988-991Crossref PubMed Scopus (129) Google Scholar). The four members of the Id protein family (Id1-Id4) function by directly associating with and modulating the activity of several families of transcriptional regulators (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 10Lasorella A. Noseda M. Beyna M. Yokota Y. Iavarone A. Nature. 2000; 407: 592-598Crossref PubMed Scopus (430) Google Scholar, 11Yates P.R. Atherton G.T. Deed R.W. Norton J.D. Sharrocks A.D. EMBO J. 1999; 18: 968-976Crossref PubMed Scopus (128) Google Scholar, 12Roberts C.E. Deed R.W. Inoue T. Norton J.D. Sharrocks A.D. Mol. Cell Biol. 2001; 21: 524-533Crossref PubMed Scopus (118) Google Scholar). However, compelling biochemical and genetic data implicate members of the ubiquitously expressed Class A, basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; HLH, helix-loop-helix; FRAP, fluorescence recovery after photobleaching; FLIP, fluorescence loss in photobleaching; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent protein; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; FP, fluorescent protein; ROI, region of interest; Pipes, 1,4-piperazinediethanesulfonic acid.1The abbreviations used are: bHLH, basic helix-loop-helix; HLH, helix-loop-helix; FRAP, fluorescence recovery after photobleaching; FLIP, fluorescence loss in photobleaching; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent protein; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; FP, fluorescent protein; ROI, region of interest; Pipes, 1,4-piperazinediethanesulfonic acid. family of “E proteins” as the most important heterodimerization targets for Id proteins in the coordinate regulation of gene expression during cell fate determination (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 2Benezra R. Oncogene. 2001; 20: 8288-8289Crossref Google Scholar, 13Benezra R. Davis R. Lockshon D. Turner D. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar, 14Massari M. Murre C. Mol. Cell Biol. 2000; 20: 429-440Crossref PubMed Scopus (1352) Google Scholar). In mammals, there are three E protein family genes, E2A (encoding three alternatively spliced variants, E12, E47, and E2-5), E2-2 (ITF2), and HEB (14Massari M. Murre C. Mol. Cell Biol. 2000; 20: 429-440Crossref PubMed Scopus (1352) Google Scholar). The E proteins bind to a consensus “E-box” recognition sequence, present in the transcriptional control regions of numerous cellular genes, either as a homodimer or, more commonly, as a heterodimeric partner with a member of the much larger family of tissue-specific, Class B bHLH proteins. Id proteins lack a basic, DNA-binding domain, and they heterodimerize avidly (via their HLH domain) with bHLH E proteins (13Benezra R. Davis R. Lockshon D. Turner D. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar) to prevent the latter from binding to DNA (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 14Massari M. Murre C. Mol. Cell Biol. 2000; 20: 429-440Crossref PubMed Scopus (1352) Google Scholar). Since E proteins are obligate heterodimerization partners for tissue-specific bHLH proteins, this provides a common mechanism of negative regulation of transcription underlying cell fate decisions in multiple lineages (2Benezra R. Oncogene. 2001; 20: 8288-8289Crossref Google Scholar). Id proteins readily associate with and inhibit the DNA binding function of bHLH proteins in vitro and will inhibit their E-box-dependent gene regulatory and biological functions in vivo (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 2Benezra R. Oncogene. 2001; 20: 8288-8289Crossref Google Scholar). However, essentially nothing is known about how the opposing functions of Id proteins and their heterodimerization target bHLH E proteins are integrated at the level of the subcellular architecture. In particular, it is unclear precisely how, in a physiological context, Id proteins are able to overcome the large DNA-binding free energy of bHLH-bHLH homo-/heterodimers in order to sequester them as Id-bHLH heterodimers. To address these issues, we have used fluorescence microscopy techniques (fluorescence recovery after photo-bleaching (FRAP), fluorescence loss in photobleaching (FLIP), and fluorescence resonance energy transfer (FRET)) (15Lippincott-Schwartz J. Snapp E. Kenworthy A. Nat. Rev. Mol. Cell Biol. 2001; 2: 444-456Crossref PubMed Scopus (962) Google Scholar) with constructs expressing proteins tagged with either enhanced yellow fluorescent protein (EYFP) or enhanced cyan fluorescent protein (ECFP) to study “real time” diffusion dynamics and heterodimeric interactions of Id and bHLH protein in single living cells. Our results show that bHLH protein is localized within distinct chromatin-associated foci, where it exists in dynamic equilibrium between the DNA-bound and non-DNA-bound state. Id proteins disrupt this chromatin compartmentalization of bHLH proteins. This suggests that Id proteins act by sequestering pools of transiently diffusing nucleoplasmic bHLH protein and preventing reassociation with chromatin domains. This would provide a mechanism through which Id proteins could overcome the large DNA-binding free energy of bHLH proteins without stabilization of Id-bHLH heterodimers by higher order oligomeric structure and thereby function as potent transcription factor antagonists. DNA Constructs—cDNA constructs encoding full-length Id3 and E47 proteins (inserted in the vector, pcDNA3; Invitrogen) have been described previously (16Wilson J.W. Deed R.W. Inoue T. Balzi M. Becciolini A. Faraoni P. Potten C.S. Norton J.D. Cancer Res. 2001; 61: 8803-8810PubMed Google Scholar, 17Deed R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Constructs encoding fusions of Id3 and E47 with the enhanced green fluorescence protein variants, EYFP and ECFP, were generated by joining the coding regions at the N and C termini of the fluorescent protein moiety, in frame, onto the C- or N-terminal coding region of the Id3 or E47 genes. A mutant version of E47 that is defective in DNA binding (E47-EYFP-bm) was generated by mutating six amino acid residues in the basic, DNA-binding domain of full-length E47 as described previously (16Wilson J.W. Deed R.W. Inoue T. Balzi M. Becciolini A. Faraoni P. Potten C.S. Norton J.D. Cancer Res. 2001; 61: 8803-8810PubMed Google Scholar). The ΔN-E47 construct was generating by deleting the coding sequence from residue 1 to 475, leaving the DNA-binding and HLH regions intact. To construct the E-box-Luc reporter plasmid (pT81-4xE), synthetic double-stranded oligonucleotides containing four tandem repeats of the E-box sequence (upper strand, 5′-CAACACCTGCTGCCTCCCAACACCTGCTGCCTCCCAACACCTGCTGCCTCCCAACACCTGCC-3′) were ligated to the BamHI/XhoI sites upstream of the thymidine kinase minimal promoter of the pT81 plasmid (12Roberts C.E. Deed R.W. Inoue T. Norton J.D. Sharrocks A.D. Mol. Cell Biol. 2001; 21: 524-533Crossref PubMed Scopus (118) Google Scholar). DNA construction and PCR mutagenesis was performed by standard recombinant DNA procedures. Cell Culture and DNA Transfection—Human embryonic kidney epithelial cells (293) were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum either on glass cover slips, in plastic flasks, or in 12-well plates. DNA-mediated transfection was performed by the standard calcium phosphate method as described previously (16Wilson J.W. Deed R.W. Inoue T. Balzi M. Becciolini A. Faraoni P. Potten C.S. Norton J.D. Cancer Res. 2001; 61: 8803-8810PubMed Google Scholar). For reporter gene assays, cells were transfected in 12-well plates with a total of 6.0 μg of DNA. DNA inputs were normalized with the addition of empty vector, pcDNA3. Cells were harvested 48 h after transfection, and preparation of extracts and the luciferase assay were performed using the dual luciferase reporter assay (Promega), according to the manufacturer's instructions. For normalization of transfection efficiencies, Renilla luciferase expression plasmid (pRL-TK; Promega) was included in the transfections. In some experiments, cells were lysed in situ on glass cover slips (18Mancini M.G. Liu B. J. Cell. 1999; PubMed Scopus Google Scholar) using Pipes, For of E47, cells were in and using by with R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). microscopy was performed using a to an with a EYFP was with the and was a was with the and was a were to prevent of the proteins were used for A single was a in a region of and were at either or time was performed using the at and were using more cells and transfected cells were used for control in the were to and of the fluorescence at time these data were by a single to the time constant was using data from cells were for was also performed by fluorescence microscopy using a as described previously with the by an S. M. H. 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PubMed Scopus Google Scholar) was using the DNA of was performed using a E47 and Id3 bHLH E protein, E47, and its potent Id have been at a and cellular level as a for Id-bHLH interactions (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar). Id3 will also inhibit gene regulatory and biological of E proteins in heterodimeric with Class B bHLH proteins, by G. H. Deed R. Norton J.D. Cell 1996; Google Scholar). the structures of fluorescent proteins generated by the coding regions of the EYFP and onto the and residue of E47 and onto the residue of Since the of the fluorescent protein was with the of proteins, for Id3 amino acid residues for Id3 residues for we constructs for In in vitro assays, and fusions of E47 to an E-box as expected (16Wilson J.W. Deed R.W. Inoue T. Balzi M. Becciolini A. Faraoni P. Potten C.S. Norton J.D. 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In order to sequester bHLH proteins as Id-bHLH Id proteins able to overcome the large DNA-binding free energy of bHLH proteins. on reassociation studies synthetic in vitro show that Id-bHLH are by a higher order oligomeric it been that Id-bHLH is through of higher order structures R. J.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is that we not any for a higher order oligomeric of Id-bHLH heterodimers in cells that would explain the of Id proteins to overcome the large DNA-binding free energy of bHLH proteins, as in vitro. Id proteins lack a basic, DNA-binding domain, they are to function as negative of bHLH proteins through a mechanism of DNA-binding activity the of Id-bHLH (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 2Benezra R. Oncogene. 2001; 20: 8288-8289Crossref Google Scholar, 13Benezra R. Davis R. Lockshon D. Turner D. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1785) Google Scholar, 14Massari M. Murre C. Mol. Cell Biol. 2000; 20: 429-440Crossref PubMed Scopus (1352) Google Scholar). However, studies of proteins in cells show that of DNA binding not for the of Id proteins. In common with E47, a DNA E47 mutant localized to as an in larger could also freely diffusible by with Id The that Id protein the compartmentalization of bHLH proteins a of Id protein function that also explain the of these proteins to overcome the DNA-binding free energy of bHLH proteins without stabilization of Id-bHLH heterodimers by higher order By sequestering transiently diffusing nucleoplasmic bHLH protein Id-bHLH heterodimers that are from chromatin Id proteins would able to prevent DNA binding and thereby accomplish a of bHLH transcription factor would of in studies to the of this directly in vivo by using a in vitro Id proteins the of transcriptional that function by negative of DNA some members of the bHLH S. S. Y. N. Yokota Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, N. K. T. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), D. PubMed Scopus Google Scholar), and M.G. Nature. PubMed Scopus Google Scholar) protein The in which the function of Id proteins is integrated at the level of the as in the present study also to these transcription factor in a mechanism through which they overcome the DNA-binding free energy of their target proteins. M. and M. T. for T. for the and C. for