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

Abstract

At the G₁/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 G₁/S phase transition.

ACKNOWLEDGMENTS

We thank John Leszyk (University of Massachusetts Protein Microsequencing Facility) for expert advice and assistance with MALDI-TOF analysis and peptide sequencing. We thank the National Cell Culture Center for providing HeLa cell cultures. We also thank Martin Montecino, Corey Braastad, Mai Luong, Caroline van der Meijden, Angela Miele, and Patricia Sue Vaughan for stimulating discussions and/or experimental assistance.

This study was supported by National Institutes of Health grant GM32010.

R.-L.X. and R.M. contributed equally to this work.

The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.