Identification of a 10-base pair protein binding site in the promoter of the hamster H3.2 gene required for the S phase dependent increase in transcription and its interaction with a Jun-like nuclear factor.

The hamster histone H3.2 promoter contains an AP-1-like element (referred to as site X) that contains the sequence CGAGTCA. This site differs from the Jun/AP-1 consensus sequence by one base and is also similar to the cyclic AMP response element. Similar AP-1/cyclic AMP response element-like sites have been found in the promoters of other histone H3 genes and are known to bind proteins either in vivo or in vitro. Using site directed mutagenesis, we demonstrate that a 10-base pair region which encompasses site X is a positive control element that is necessary for the S phase dependent increase in H3.2 transcription in cells synchronized by serum stimulation or aphidicolin block. DNase I footprint analysis shows that mutating site X eliminates v-Jun and hamster cellular factor(s) binding. Further in vitro analysis with gel retardation assays reveals that the flanking sequence of this site is necessary for the formation of an H3.2 specific complex that can be distinguished from complexes formed with a collagenase or SV40 AP-1 element. Antibodies specific to the different members of the Jun and Fos family of transcription factors show that, in gel retardation assays, a Jun-like factor is a component of the H3.2 specific complex. However, the H3.2 specific complex exhibits different reactivity toward the Jun and Fos specific antibodies as compared to complexes formed with a collagenase AP-1 element. We hypothesize that a unique protein complex, containing a component related to the AP-1 family of transcription factors, binds to the AP-1-like motif of the hamster H3.2 promoter and may be involved in the S phase dependent regulation of transcription.