Transcriptional element H4‐site II of cell cycle regulated human H4 histone genes is a multipartite protein/DNA interaction site for factors HiNF‐D, HiNF‐M, and HiNF‐P: Involvement of phosphorylation

Abstract

Cell cycle regulated gene expression was studied by analyzing protein/DNA interactions occurring at the H4-Site II transcriptional element of H4 histone genes using several approaches. We show that this key proximal promoter element interacts with at least three distinct sequence-specific DNA binding activities, designated HiNF-D, HiNF-M, and HiNF-P. HiNF-D binds to an extended series of nucleotides, whereas HiNF-M and HiNF-P recognize sequences internal to the HiNF-D binding domain. Gel retardation assays show that HiNF-D and HiNF-M each are represented by two distinct protein/DNA complexes involving the same DNA binding activity. These results suggest that these factors are subject to post-translational modifications. Dephosphorylation experiments in vitro suggest that both electrophoretic mobility and DNA binding activities of HiNF-D and HiNF-M are sensitive to phosphatase activity. We deduce that these factors may require a basal level of phosphorylation for sequence specific binding to H4-Site II and may represent phosphoproteins occurring in putative hyper- and hypo-phosphorylated forms. Based on dramatic fluctuations in the ratio of the two distinct HiNF-D species both during hepatic development and the cell cycle in normal diploid cells, we postulate that this modification of HiNF-D is related to the cell cycle. However, in several tumor-derived and transformed cell types the putative hyperphosphorylated form of HiNF-D is constitutively present. These data suggest that deregulation of a phosphatase-sensitive post-translational modification required for HiNF-D binding is a molecular event that reflects abrogation of a mechanism controlling cell proliferation. Thus, phosphorylation and dephosphorylation of histone promoter factors may provide a basis for modulation of protein/DNA interactions and H4 histone gene transcription during the cell cycle and at the onset of quiescence and differentiation.

Coordinate Control and Selective Expression of the Full Complement of Replication-dependent Histone H4 Genes in Normal and Cancer Cells

“…Thus, Site II cell cycle regulatory mechanisms at the onset of S phase function independently of E2F (2,15,18). Three histone nuclear factors (HiNF-M, -D, and -P) 3 interact with Site II to mediate cell cycle control of transcription at the onset of S phase (4,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Recently, HiNF-P has been identified as the protein that, in conjunction with the cyclin E/CDK2-responsive protein p220…”

Transcriptional activation of the histone nuclear factor P (HiNF-P) gene by HiNF-P and its cyclin E/CDK2 responsive co-factor p220NPAT defines a novel autoregulatory loop at the G1/S phase transition

“…SP1 regulates housekeeping genes under control of TATA-less promoters (Suske 1999), as well as cell cycle related genes (e.g., histone H4 and CDK2) Xie et al, 2003). Control of the HiNF-P gene promoter by SP1 may account at least in part for the proliferation-specific expression of HiNF-P that we have observed in many cell types (Aziz et al, 1998;Gruol and Altschmied 1993;Hovhannisyan et al, 2003;Mitra et al, 2003;Shakoori et al, 1995;van der Meijden et al, 1998;van Wijnen et al, 1991). We also find that the E2F1 represses the HiNF-P gene in SAOS-2 cells.…”

“…The DNA binding activity of HiNF-P that supports its interaction with the genomic loci of histone H4 genes in vivo Pauli et al, 1987) is specific for a broad spectrum of proliferating cells (Aziz et al, 1998;Mitra et al, 2003;Shakoori et al, 1995;van den Ent et al, 1993;van Wijnen et al, 1991;van Wijnen et al, 1992). HiNF-P protein levels and in vivo occupancy of its site are constitutive during the cell cycle (Medina et al, 2006;Pauli et al, 1987), but both DNA binding activity and protein levels of HiNF-P are down regulated below the level of detection upon differentiation of HL60 cells .…”

“…HiNF-P recognizes an unusually large consensus sequence within histone H4 genes (Aziz et al, 1998;Holmes et al, 2005;Stein et al, 1991;van Wijnen et al, 1991;van Wijnen et al, 1992) that links H4 gene transcription to the Cyclin E/CDK2/p220 NPAT /HiNF-P pathway. HiNF-P is related to H4TF-2, a DNA binding protein that was not characterized beyond its molecular mass (Dailey et al, 1988;Mitra et al, 2003;van Wijnen et al, 1992), and was serendipitously re-discovered as methyl CpG Binding Protein 2 (MBD2) Zn-finger Interacting Factor (MZIF) in yeast two hybrid assays (Sekimata et al, 2001;Sekimata and Homma 2004).…”

“…HiNF-P DNA binding activity has been detected in a broad range of cells and tissues from different mammalian species (Aziz et al, 1998;Langdahl et al, 1997;Shakoori et al, 1995;van den Ent et al, 1993;van der Meijden et al, 1998;van Wijnen et al, 1991;van Wijnen et al, 1992), and HiNF-P binding sites in the promoters of H4 genes are only occupied in cells progressing through the cell cycle, as established by genomic DNaseI footprinting Pauli et al, 1987) and chromatin immunoprecipitations (Miele et al, 2005;Mitra et al, 2003). HiNF-P (MIZF) mRNA is ubiquitously expressed in a number of tissues and cells (Sekimata et al, 2001) and robustly expressed in human embryonic stem cells (Becker et al, 2006;Becker et al, 2007).…”