Repetitive Alu elements form a cruciform structure that regulates the function of the human CD8 alpha T cell-specific enhancer.
We previously identified a T cell-specific enhancer in the last intron of the human CD8 alpha gene that is adjacent to a sequence element that significantly represses enhancer function. This negative regulatory region consists of a half-Alu sequence that has potential to base-pair with a downstream Alu element, which is part of the fully active enhancer, to form a cruciform structure. The activity of this half-Alu silencer sequence is position and orientation-dependent, suggesting that DNA structure plays an important role in its function. Using site-directed mutational analysis and P1 nuclease mapping, we directly demonstrate that formation of a cruciform structure is required for repression of enhancer function in transient transfection assays. Finally, a P1 nuclease-sensitive site is present in the endogenous CD8 alpha gene in T cell lines providing indirect evidence that the stem-loop may form in vivo. Taken together, these results suggest that Alu elements may contribute to the regulation of the CD8 alpha gene enhancer through the formation of secondary structure that disrupts enhancer function.
Hanke
J H
JH
Department of Molecular Genetics and Protein Chemistry, Pfizer Central Research, Groton, CT 06340.
Hambor
J E
JE
Kavathas
P
P
D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion / Q000502:physiology; D015719:Single-Strand Specific DNA and RNA Endonucleases
D002843:Chromatin; D004277:DNA, Single-Stranded; D015719:Single-Strand Specific DNA and RNA Endonucleases