HiNF-P Directly Links the Cyclin E/CDK2/p220^NPAT Pathway to Histone H4 Gene Regulation at the G₁/S Phase Cell Cycle Transition

Abstract

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G 1 /S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G 1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220NPAT , a substrate of cyclin E/CDK2, to coactivate histone genes during S phase. HiNF-P and p220 are targeted to, and colocalize at, subnuclear foci (Cajal bodies) in a cell cycle-dependent manner. Genetic or biochemical disruption of the HiNF-P/p220 interaction compromises histone H4 gene activation at the G 1 /S phase transition and impedes cell cycle progression. Our results show that HiNF-P and p220 form a critical regulatory module that directly links histone H4 gene expression at the G 1 /S phase transition to the cyclin E/CDK2 signaling pathway at the R point.Fidelity of genome replication in eukaryotic cells is essential for cell division and necessitates the stringent coupling of histone biosynthesis with DNA replication to ensure that nascent DNA is immediately assembled into chromatin during DNA synthesis. Cell division requires staged expression of genes in response to growth factors, which induce cell growth from quiescence or maintain competency for cell cycle progression during periods of active proliferation. Stimulation of cell proliferation initially triggers a cyclin/cyclin-dependent kinase (CDK) cascade, which activates the cyclin E/CDK2 kinase complex at the restriction (R) point (17,19). The R point is the major cell cycle checkpoint that controls the commitment for DNA replication in late G 1 via CDK2-dependent release of E2F from Rb-related proteins. The R point is mechanistically linked through E2F to activate the gene regulatory program necessary for nucleotide metabolism and DNA replication (17,19). Passage beyond the R point permits growth factor-independent entry into S phase and subsequent cell cycle stages. However, cell cycle progression remains constrained by multiple checkpoints, including surveillance mechanisms that monitor DNA integrity and fidelity of chromatin assembly.We postulate that the induction of histone gene expression at the G 1 /S phase transition represents a second necessary cell cycle regulatory event. The coupling of DNA synthesis with histone protein production is maintained by coordinately inducing expression of the multiple core histone gene subtypes, including the 15 distinct histone H4 genes, at the onset of S phase (1,3,12,20,23,24). The cell cycle regulatory sequence of histone H4 genes lacks E2F binding sites (28). We have recently identified the key transcription factor of H4 genes, histone nuclear factor P (HiNF-P), which interacts with a highly conserved histone H4 subtype-specific element in the site II cell cycle regulatory domain (16). HiNF-P supports histone gene transcription at the G 1 /S phase transition indepe...

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

“…The three rows differ in staining for cyclin A (first row), cyclin B (second row), or cyclin E (third row). Miele et al, 2005). Our results are consistent with the idea that mechanisms for synthesis and processing of histone gene transcripts are established in human embryonic stem cells and maintained in lineage-differentiated somatic cells.…”

“…The micrographs presented below the diagrams (row 2) show enlargements of the foci containing p220 NPAT and phospho-p220 NPAT that are presented in Part A (row 4). Miele et al, 2005). The prototypical Cajal body component coilin interacts with U7snRNP, thereby providing structural linkage between Cajal bodies and the histone premRNA processing machinery (Bellini and Gall, 1998).…”

“…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).…”