Transcriptional regulation of cell cycle controlled genes is fundamental to cell division in eukaryotes and a broad spectrum of physiological processes directly related to cell proliferation. Expression of the cell cycle dependent human H4, H3 and HI histone genes is coordinately regulated at both the transcriptional and post- transcriptional levels. We have systematically analyzed the protein/DNA interactions of the immediate 5' regions of three prototypical cell cycle controlled histone genes, designated H4- FOI08, H3- ST519 and HI- FNCI6, to define components of the cellular mechanisms mediating transcriptional regulation.

Multiple biochemically distinct protein/DNA interactions were characterized for each of these genes, and the binding sites of several promoter-specific nuclear DNA binding activities were delineated at single nucleotide resolution using a variety of techniques. These findings were integrated with results obtained by others and revealed that the in vitro factor binding sites in H4, H3 and H1 histone promoters coincide with genomic protein/DNA interaction sites defined in vivo for the H4- FOI08 and H3- ST519 genes, and with evolutionarily conserved cis-acting sequences shown to affect the efficiency of histone gene transcription. Specifically, we have defined binding sites for Spl, ATF, CP1/NF-Y, HiNF-D, HiNF-M, HiNF- P and HMG- I related factors. Based on sequence-similarities and cross-competition experiments, we postulate that most of these protein/DNA interaction elements are associated with more than one class of histone genes. Thus, the protein/DNA interactions characteri zed in this study may represent components of a cellular mechanism that couples transcription rates of the various histone gene classes.

Regulation of the protein/DNA interactions involved in transcriptional control of these H4, H3 and H1 histone genes was investigated in a spectrum of cell types using several distinct in vitro cell culture models for the onset of differentiation and quiescence, as well as cell cycle progression. Moreover, we studied control of histone gene associated DNA binding activities during hepatic development from fetus to adult in transgenic mice reflecting the onset of differentiation and quiescence in vivo.We show that the H4 histone promoter protein/DNA selectively interaction mediated by factor Hi NF - D is modulated, and directly at the level of DNA binding activity, during the entry into, progress through and exit from the cell cycle in normal diploid cells, as well as during hepatic development. The regulation of this prote n/DNA interaction occurs in parallel with analogous interactions occurring in H3 and H1 histone genes. Moreover, these proliferation-specific protein/DNA interactions are collectively deregulated during the cell cycle in four distinct cell types displaying properties of the transformed phenotype. Hence, the cell ul ar competency to coordinately transcribe distinct classes of histone genes during the cell cycle may be mediated by the intricate interplay of constitutively expressed general transcri pti on factors and temporally regul ated, cell growth controlled nuclear factors interacting specifically with cycle cell dependent histone genes.

Finally, we show that HiNF- D is represented by two electrophoretically distinct species. The ratio of these forms of HiNF- fluctuates dramatically during the cell cycle of normal diploid cells, but remains relatively constant in tumor cells. Total HiNF-D binding activity embodied by both HiNF-D species is negatively influenced in vitro by incubation with exogenous phosphatase activity. These observations provide a first indication for the hypothesis that HiNF-D may exist in distinct post-translationally modified forms that are subject to a stringent cell growth control mechanism involving protein ki nases and phosphatases. Such a cellular post-translational modification mechanism, which directly impinges on (or activates) the DNA binding activity of a key factor controlling histone genes, would provide a highly efficient means by which to influence the rate of transcription in rapid response to intra-cellular requirements for histone mRNA and extra-cellular cues signalling the onset and cessation of cell proliferation.