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.
O'Toole
Peter J
PJ
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
Inoue
Toshiaki
T
Emerson
Lindsay
L
Morrison
Ian E G
IE
Mackie
Alan R
AR
Cherry
Richard J
RJ
Norton
John D
JD
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry*; D019281:Dimerization; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000737:chemistry*; D011485:Protein Binding; D051820:TCF Transcription Factors; D013997:Time Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D014162:Transfection
D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051796:Inhibitor of Differentiation Proteins; D009363:Neoplasm Proteins; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C075640:ID3 protein, human; D004247:DNA