TCRalpha enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex.
The TCR alpha enhancer (Ealpha) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ealpha occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ealpha occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Ealpha is known to be active (CD4(+)CD8(+)-DP cells), but surprisingly, also in cells at an earlier developmental stage where Ealpha is not active (CD4(-)CD8(-)-DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo-protein complex topography. Stage-specific differences in factor composition at Ealpha sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre-assembled nucleo-protein complex correlate with the onset of Ealpha activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.
Spicuglia
S
S
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille and INSERM U119, 27 boulevard Leï Roure, 13009 Marseille, France.
Payet
D
D
Tripathi
R K
RK
Rameil
P
P
Verthuy
C
C
Imbert
J
J
Ferrier
P
P
Hempel
W M
WM
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002454:Cell Differentiation; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D019673:Genes, T-Cell Receptor alpha / Q000235:genetics*; D016678:Genome; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D015533:Transcriptional Activation
D015704:CD4 Antigens; D016827:CD8 Antigens; D009687:Nuclear Proteins; D009707:Nucleosomes; D013463:Sulfuric Acid Esters; D004247:DNA; C007482:dimethyl sulfate