Identification of a second conserved element within the coding sequence of a mouse H3 histone gene that interacts with nuclear factors and is necessary for normal expression

Abstract

Replication-dependent histone genes of all four nucleosomal classes are coordinately up-regulated at the beginning of S phase of the eukaryotic cell cycle. The universality and importance of this process in eukaryotic cells suggest that common regulatory mechanisms are involved in controlling the high level of expression of these histone genes. We have previously identified the α element within mouse H2a.2 and H3.2 coding region activating sequences (CRAS), which is involved in regulation of these two replication-dependent genes. Here we report the identification of a second element within the mouse histone CRAS, the Ω element. This element interacts with nuclear proteins and we present in vivo evidence that this sequence is required for normal expression. Ω nucleotides involved in interaction with nuclear proteins have been precisely mapped by means of DNase I footprinting and methylation interference assays. A naturally occurring mutation in the Ω sequence is found in a replication-independent H3.3 gene. Mutation of the H3.2 Ω element to that of the H3.3 sequence (3 nt changes) caused a 4-fold drop in in vivo expression of the H3.2 gene in stably transfected CHO cells, equalling the effect of mutation of all 7 nt of the element. By UV cross-linking we have determined the approximate molecular weight of the Ω binding protein to be 45 kDa. Finally, we identify putative Ω sequences in the coding region of mouse H2b and H4 histone genes.