Stereospecific alignment of the X and Y elements is required for major histocompatibility complex class II DRA promoter function.
The regulatory mechanisms controlling expression of the major histocompatibility complex (MHC) class II genes involve several cis-acting DNA elements, including the X and Y boxes. These two elements are conserved within all murine and human class II genes and are required for accurate and efficient transcription from MHC class II promoters. Interestingly, the distance between the X and Y elements is also evolutionarily conserved at 18 to 20 bp. To investigate the function of the invariant spacing in the human MHC class II gene, HLA-DRA, we constructed a series of spacing mutants which alters the distance between the X and Y elements by integral and half-integral turns of the DNA helix. Transient transfection of the spacing constructs into Raji cells revealed that inserting integral turns of the DNA helix (+20 and +10 bp) did not reduce promoter activity, while inserting or deleting half-integral turns of the DNA helix (+15, +5, and -5 bp) drastically reduced promoter activity. The loss of promoter function in these half-integral turn constructs was due neither to the inability of the X and Y elements to bind proteins nor to improper binding of the X- and Y-box-binding proteins. These data indicate that the X and Y elements must be aligned on the same side of the DNA helix to ensure normal function. This requirement for stereospecific alignment strongly suggests that the X- and Y-box-binding proteins either interact directly or are components of a larger transcription complex which assembles on one face of the DNA double helix.
Vilen
B J
BJ
Lineberger Cancer Research Center, Chapel Hill, North Carolina.
Cogswell
J P
JP
Ting
J P
JP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D004273:DNA, Neoplasm / Q000235:genetics; D005802:Genes, MHC Class II; D006801:Humans; D008745:Methylation; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013237:Stereoisomerism; D014162:Transfection
D004273:DNA, Neoplasm; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase