THE INTEGRATED ACTIVITIES OF IRF-2 (HINF-M), CDP CUT (HINF-D) AND H4TF-2 (HINF-P) REGULATE TRANSCRIPTION OF A CELL-CYCLE CONTROLLED HUMAN HISTONE H4 GENE - MECHANISTIC DIFFERENCES BETWEEN DISTINCT H4 GENES/

Maximal transcription of a prototypical cell cycle controlled histone H4 gene requires a proliferation-specific in vivo genomic protein/DNA interaction element, Site II. Three sequence-specific transcription fa ctors interact with overlapping recognition motifs within Site II: int erferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-spec ific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, nat ural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functio nal consequences of these variations have not been investigated. To ad dress the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis o f Site LT transcriptional motifs. These mutants were tested for abilit y to bind each of the Site LT cognate proteins, and subsequently evalu ated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also ana lyzed the effect of over-expressing IRF-2 on CAT reporter gene express ion driven by mutant H4 promoters and assessed H4 transcriptional cont rol in cells nullizygous for IRF-1 and IRF-2. Our results show that th e recognition sequence for IRF-2 (HiNF-M) is the dominant component of Site II and modulates H4 gene transcription levels by 3 fold. However , the overlapping recognition sequences for IRF-2 (HiNF-M), H4TF-2 (Hi NF-P) and CDP/cut (HiNF-D) together-modulate H4 gene transcription lev els by at least an order of magnitude. Thus, maximal activation of H3 gene transcription during the cell cycle in vivo requires the integrat ed activities of multiple transcription factors at Site II. We postula te that the composite organization of Site LT supports responsiveness to multiple signalling pathways modulating the activities of H4 gene t ranscription factors during the cell cycle. Variations in Site II sequ ences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic pro perties.