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Identify and characterize regulatory DNA elements responsible for silencing human CD4 gene expression in specific T-cell lineages.

Identification and characterization of a human CD4 silencer

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Identify and characterize regulatory DNA elements responsible for silencing human CD4 gene expression in specific T-cell lineages.
Screened transgenic mice carrying human CD4 gene fragments to localize silencer activity to a 484-bp intronic segment, further narrowed to 190 bp.
Identified protein-binding elements using deoxyribonuclease I (DNase I) footprinting, designated FP I and FP II.
Evaluated regulatory activity and DNA-protein interactions via transient transfection assays and electrophoretic mobility shift assays (EMSA) in CD4 and CD8 T cells, including base-substitution mutagenesis.
Isolated a 190-bp lineage-specific silencer region in the first intron of the human CD4 gene containing two distinct DNase I footprint binding sites.
Transient transfection demonstrated that FP I and FP II drive strong silencing restricted to CD8 cells, whereas FP II functions as an enhancer and FP I has no effect in CD4 cells.
Base substitutions in FP I abolished CD8-restricted silencing activity as well as protein-DNA complex formation in EMSA, despite equivalent baseline binding in CD4 and CD8 cells.

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Experimental study uncovers an intronic silencer regulating CD4 expression in T cells, suggesting a core mechanism governing lineage commitment.

Donda et al. (1996) studied this question.

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Experimental study uncovers an intronic silencer regulating CD4 expression in T cells, suggesting a core mechanism governing lineage commitment.