Multiple Mechanisms Regulate the Proliferation-Specific Histone Gene Transcription Factor HiNF-D in Normal Human Diploid Fibroblasts

The proliferation-specific transcription factor complex HiNF-D interacts with sequence specificity in a proximal promoter element of the human H4 histone gene FO108, designated Site II. The occupancy of Site II by HiNF-D has been implicated in proper transcription initiation and as a component of the cell cycle regulation of this gene. In the present study we have investigated the role of the HiNF-D/Site II interaction in controlling the level of H4 histone gene transcription during modifications of normal cellular growth. HiNF-D binding activity is present at high levels in rapidly proliferating cultures of human diploid fibroblasts and is reduced to less than 2% upon the cessation of proliferation induced by serum deprivation of sparsely populated fibroblast cultures. Density-dependent quiescence also abolishes HiNF-D binding activity. Downregulation of transcription from the H4 gene occurs concomitant with the loss of the HiNF-D/Site II interaction, further suggesting a functional relationship between Site II occupancy and the capacity for transcription. Serum stimulation of quiescent preconfluent cells results in an increase in HiNF-D binding activity as the cells are resuming DNA synthesis and H4 histone gene transcription. Density-inhibited quiescent cells respond to serum stimulation with only a minimal increase in the HiNF-D binding activity, 30% of maximal levels. However, H4 histone gene transcription is stimulated to a level equal to that detected in extracts of the sparsely populated serum-stimulated cultures. These results suggest that there is a threshold level of HiNF-D binding activity necessary for the activation of H4 histone gene transcription. Additionally, these findings suggest that there may be a mechanism repressing HiNF-D binding activity in the density-inhibited cultures which is not operative in the sparsely populated, serum-deprived cultures. Density-inhibited cultures may have reached a state analogous to the initial steps of differentiation and have invoked a series of mechanisms to decrease expression of proliferation-specific factors. Serum stimulation is able to overcome the one mechanism downregulating HiNF-D in both sparsely populated and density-inhibited quiescent cultures but is unable to reverse the repression of proliferation-specific factors that occurs in density-inhibited cultures. These results are consistent with the presence of at least two levels of control over the HiNF-D/Site II interaction which are responsive to and reflect the proliferative state of the cell and the extent to which the cell exhibits properties associated with differentiation.