Identification of HiNF-P, a Key Activator of Cell Cycle-Controlled Histone H4 Genes at the Onset of S Phase

Abstract

At the G 1 /S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G 1 /S phase transition.The G 1 /S phase transition represents the critical stage during the somatic cell cycle that defines cellular commitment to replicate the genome and to progress towards mitotic division. Passage beyond the G 1 /S boundary depends initially on the activation of a cyclin/cyclin-dependent kinase (CDK) cascade by growth factors and induction of the cyclin E/CDK2 kinase complex at the restriction (R) point (9,14,29). Subsequently, at the onset of S phase, de novo synthesis of histone proteins is required to package nascent DNA into chromatin immediately upon initiation of DNA synthesis (28,35,36). The exquisitely stringent coupling between histone biosynthesis and DNA replication necessitates the transcriptional activation of the 14 distinct human genes encoding histone H4, the most highly conserved nucleosomal protein (1,13,19,28).Control of histone genes provides a paradigm for gene expression that is temporally and functionally linked to DNA synthesis. It has long been postulated but not yet experimentally validated that the multiple histone H4 genes are coordinately regulated by a single histone H4 gene subtype-specific factor. However, the identity of this protein has never been established. We and others have previously demonstrated that histone H4 genes are regulated by multiple elements and cognate DNA binding activities (6,7,16,30,31,44). The histone gene proximal promoter element site II interacts with three factors (HiNF-M, -D, and -P) and mediates cell cycle control of transcription at the onset of S phase (3,6,7,16,30,31,36,(43)(44)(45)(46)(47). Site II encompasses the H4 subtype-specific element that is phylogenetically conserved among multiple histone H4 genes in metazoan species. The cell cycle regulatory mechanisms operative at site II at the onset of S phase function independently of...

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

“…One principal event during the somatic cell cycle is the induction of the HiNF-P/ p220 NPAT co-activation complex by CDK dependent phosphorylation of p220 NPAT (Zhao et al, 1998(Zhao et al, , 2000Ma et al, 2000;Mitra et al, 2003;Wei et al, 2003;Ye et al, 2003;Miele et al, 2005). Here we examine the appearance of subnuclear foci containing p220 NPAT and phosphorylation of p220 NPAT in ES cells to understand the regulation of the G1/S phase transition relative to somatic cells.…”

“…Seminal studies by Pardee and colleagues have defined the restriction (R) point in the G1 phase of somatic cells that represents the stage when commitment towards initiation of DNA synthesis at the G1/S phase transition becomes growth factor independent (Pardee, 1974(Pardee, , 1989. Studies in our laboratory have examined the functional coupling between the R point and signaling pathways that control the activation of histone gene expression ('S point') which is essential for the packaging of newly replicated DNA (Stein et al, 1992(Stein et al, , 1996Luong et al, 2002;Mitra et al, 2003;Braastad et al, 2004;Holmes et al, 2005;Miele et al, 2005;Becker et al, 2006;Becker et al, 2007). One essential difference between human ES and somatic cells is the length of G1 phase during which cells commit to S phase entry.…”

“…Similar to somatic cells, ES cells express histone gene regulatory factors (e.g., HiNF-P and p220 NPAT ) and exhibit S phase specific expression of distinct members of the DNA replication dependent histone gene family (Becker et al, 2006(Becker et al, , 2007. In somatic cells, critical events for the onset of S phase include the cell cycle dependent phosphorylation of p220 NPAT by cyclin E/CDK2 at nuclear foci related to Cajal bodies and the subsequent recruitment of p220 NPAT by transcription factor HiNF-P to histone H4 gene promoters (Zhao et al, 1998(Zhao et al, , 2000Ma et al, 2000;Mitra et al, 2003;Wei et al, 2003;Ye et al, 2003;Miele et al, 2005).…”

Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells

“…For example, recruitment of the coactivator protein p220 NPAT (nuclear protein, ataxia-telangiectasia locus) by transcription factor HiNF-P (histone nuclear factor-P) to histone H4 gene promoters, as well as cell cycle-dependent phosphorylation of p220 NPAT by cyclin E/CDK2 to induce histone gene transcription occur at these intranuclear sites (7,8,(13)(14)(15)(16)(17). Newly synthesized histone transcripts are not polyadenylated, and their cleavage requires a U7 small nuclear ribonucleoprotein complex (U7 snRNP) that contains U7 snRNA and the protein subunits Lsm10 (U7 snRNP-specific Sm-like protein LSM10) and Lsm11 (U7 snRNP-specific Sm-like protein LSM11), whereas a specific RNA hairpin in histone transcripts interacts with stem loop binding protein (SLBP) (18)(19)(20)(21)(22).…”

“…However, hES cells lack a classical R point and have the capacity for continuous cell division. A principal mechanism that is required for the initiation of S phase in hES cells is the induction of histone gene expression, which is essential for the packaging of newly replicated DNA into chromatin by specific transcription factors (1,2,(5)(6)(7)(8)(9)(10)(11)(12).…”