Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro.
The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain was sufficient to form a higher-order complex with AML1/Ets-1, but exhibited only partial activity in transcription. We conclude that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCR alpha enhancer in a chromatin environment.
Mayall
T P
TP
The Salk Institute for Biological Studies, La Jolla, California 92037-1099, USA.
Sheridan
P L
PL
Montminy
M R
MR
Jones
K A
KA
D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism*; D050676:Core Binding Factor Alpha 2 Subunit; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D051821:Lymphoid Enhancer-Binding Factor 1; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism*
D002843:Chromatin; D050676:Core Binding Factor Alpha 2 Subunit; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D006609:High Mobility Group Proteins; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; C493728:RUNX1 protein, human; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014157:Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases