Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y1 14F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y1 14F alleles (PCNA(F/F)) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y1 14F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA(F/F) MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA(F/F) mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation. (C) 2012 Elsevier Inc. All rights reserved.