The Chromatin-Associated Phf12 Protein Maintains Nucleolar Integrity and Prevents Premature Cellular Senescence

ABSTRACT

Pf1, also known as Phf12 (plant homeodomain [PHD] zinc finger protein 12), is a member of the PHD zinc finger family of proteins. Pf1 associates with a chromatin-interacting protein complex comprised of MRG15, Sin3B, and histone deacetylase 1 (HDAC1) that functions as a transcriptional modulator. The biological function of Pf1 remains largely elusive. We undertook the generation of Pf1 knockout mice to elucidate its physiological role. We demonstrate that Pf1 is required for mid- to late gestation viability. Pf1 inactivation impairs the proliferative potential of mouse embryonic fibroblasts (MEFs) and is associated with a significant decrease in bromodeoxyuridine incorporation; an increase in senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence; and elevated levels of phosphorylated H2AX (γ-H2A.X), a marker associated with DNA double-strand breaks. Analysis of transcripts differentially expressed in wild-type and Pf1-deficient cells revealed the impact of Pf1 in multiple regulatory arms of the ribosome biogenesis pathways. Strikingly, assessment of the morphology of the nucleoli exposed an abnormal nucleolar structure in Pf1-deficient cells. Finally, proteomic analysis of the Pf1-interacting complexes highlighted proteins involved in ribosome biogenesis. Taken together, our data reveal an unsuspected function for the Pf1-associated chromatin complex in the ribosomal biogenesis and senescence pathways.

KEYWORDS:

• transcription
• ribosome
• Pf1
• senescence
• nucleolus

ACKNOWLEDGMENTS

We thank the staff of NYU Langone School of Medicine, B. Ueberheide, and the Proteomics Resource Center for assistance with the proteomics experiments, A. Heguy and the Genome Technology Center for assistance with the microarray experiments, and Esthelle Hoedt for help with proteomics data analyses. We also thank Beatriz Fontoura, John Goodier, Daisuke Izawa, Stefan Muller, and Celine Verheggen for the generous gift of reagents.

The work reported here was supported by the National Institutes of Health (R01CA148639 and R21CA155736 to G.D.), the Samuel Waxman Cancer Research Foundation (to G.D.), and a Feinberg NYU individual grant (to G.D.). K.M. was supported by departmental training grant 5-T32CA9161-40. The mass spectrometric experiments were in part supported by Cancer Center support grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. The Proteomics Resource Center and the Genome Technology Center are supported in part by NIH/NCI support grant P30CA016087.