The Inverted CCAAT Motif is an Indispensable Element of the Enhancer B of the Mouse Major Histocompatibility I H2-K b Gene

Abstract

We have identified a strong binding of nuclear proteins derived from Ltk^- fibroblasts to the enhancer B of the mouse MHC class I H2-K^b gene. The inverted CCAAT motif and its adjacent upstream sequences have been revealed as protein-binding sites by electrophoretic mobility-shift, methylation interference, and DNase I footprint assays. Specific mutations in the inverted CCAAT motif as well as in the 5′-flanking cytosine pentanucleotide abrogated the formation of the major DNA-protein complex. Transcription of the chloramphenicol acetyltransferase (CAT) reporter gene driven by the H2-K^b promoter in the Ltk^- cell line was reduced substantially when a two-nucleotide mutation was introduced into the CCAAT element (CCAATCgcAT). The indicated two-nucleotide mutation decreased transcription initiated from both the homologous and a heterologous promoter. Furthermore, cotransfected MHC class II transactivator (CIITA) elevated the transcription of the reporter gene under the control of the H2-K^b upstream sequences in the NIH 3T3 cell line. The intact enhancer B involving both the inverted CCAAT motif and the site α was found to play an indispensable role in the CIITA-mediated gene transactivation. The band-shift assay with the enhancer B probe revealed forming of a protein complex in a cooperative manner, which was again prevented by mutations in either element. Our results suggest an essential role of the inverted CCAAT element in the constitutive as well as inducible transcription of the mouse MHC class I genes.