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 factors interact with overlapping recognition motifs within Site II: interferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-specific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, natural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functional consequences of these variations have not been investigated. To address the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis of Site II transcriptional motifs. These mutants were tested for ability to bind each of the Site II cognate proteins, and subsequently evaluated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also analyzed the effect of over-expressing IRF-2 on CAT reporter gene expression driven by mutant H4 promoters and assessed H4 transcriptional control in cells nullizygous for IRF-1 and IRF-2. Our results show that the 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 (HiNF-P) and CDP/cut (HiNF-D) together modulate H4 gene transcription levels by at least an order of magnitude. Thus, maximal activation of H4 gene transcription during the cell cycle in vivo requires the integrated activities of multiple transcription factors at Site II. We postulate that the composite organization of Site II supports responsiveness to multiple signalling pathways modulating the activities of H4 gene transcription factors during the cell cycle. Variations in Site II sequences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic properties.

The human histone H4 gene FO108 is regulated during the cell cycle with a peak in transcription during early S phase. The cell-cycle element (CCE) required for H4 histone activation is a sequence of 11 base pairs that binds a protein factor in electrophoretic mobility shift assays that has been designated histone nuclear factor M (HiNF-M). Here we report the purification of HiNF-M, and show it to be a protein of relative molecular mass (M(r)) 48K that is identical to interferon (IFN) regulatory factor 2 (IRF-2), a negative transcriptional regulator of the IFN response. Recombinant IRF-2 (as well as the related protein IRF-1 (ref. 5)) binds the CCE specifically and activates transcription of this H4 histone gene. IRF-2 has been shown to have oncogenic potential, and our results demonstrate a link between IRF-2 and a gene that is functionally coupled to DNA replication and cell-cycle progression at the G1/S phase transition.

Cell cycle-controlled human histone genes are coordinately expressed during S phase, and transcriptional regulation involves a series of trans-acting factors (HiNFs). The proliferation-specific factor HiNF-D interacts with multiple recognition motifs in histone H4, H3, and H1 promoters. Using gel shift immunoassays, we show that CDC2, cyclin A, and an RB-related protein are ubiquitous subunits of HiNF-D binding activity isolated from several cell types. HiNF-D levels in vivo are sensitive to okadaic acid and staurosporine, indicating that HiNF-D activity and/or assembly is influenced by phosphorylation status. Thus, HiNF-D appears to be a multicomponent phosphoprotein that participates in coordinate control of multiple histone H4, H3, and H1 genes during the cell cycle. The presence of cell cycle mediators in the HiNF-D complex suggests linkage between transcriptional control of histones, enzymes involved in DNA synthesis, and the onset of DNA replication during the G1/S phase transition.

Upstream sequences of the H4 histone gene FO108 located between nt -418 to -213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4-Site III) that binds factor H4UA-1. Based on methylation interference, copper-phenanthroline protection, and competition assays, we show that H4UA-1 interacts with sequences between nt -345 to -332 containing an element displaying sequence-similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA-1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase-treatment of nuclear proteins inhibits formation of the H4UA-1 protein/DNA complex, although a complex with higher mobility (H4UA-1b) can be detected; both complexes share identical protein-DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4-Site III protein/DNA interactions by directly altering protein/protein associations. H4-Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA-1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA-1/H4-Site III interaction, concomitant with an increase of the H4UA-1b/H4-Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA-1, whereas H4UA-1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA-1 and H4UA-1b binding activities may reflect deregulation of H4-Site III interactions in transformed cells. We propose that H4-Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4-FO108 histone gene transcription.