Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro

Genes Dev. 1997 Apr 1;11(7):887-99. doi: 10.1101/gad.11.7.887.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Chromatin / metabolism*
  • Core Binding Factor Alpha 2 Subunit
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • DNA-Binding Proteins / metabolism*
  • Enhancer Elements, Genetic*
  • Gene Expression Regulation, Developmental*
  • High Mobility Group Proteins / metabolism
  • Humans
  • Jurkat Cells
  • Lymphoid Enhancer-Binding Factor 1
  • Models, Genetic
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Binding
  • Proto-Oncogene Protein c-ets-1
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-ets
  • Receptors, Antigen, T-Cell, alpha-beta / genetics*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / physiology*
  • Transcription Factors / metabolism*

Substances

  • Chromatin
  • Core Binding Factor Alpha 2 Subunit
  • Cyclic AMP Response Element-Binding Protein
  • DNA-Binding Proteins
  • ETS1 protein, human
  • High Mobility Group Proteins
  • LEF1 protein, human
  • Lymphoid Enhancer-Binding Factor 1
  • Proto-Oncogene Protein c-ets-1
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-ets
  • RUNX1 protein, human
  • Receptors, Antigen, T-Cell, alpha-beta
  • Transcription Factors
  • Cyclic AMP-Dependent Protein Kinases