Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes

Chromatin-associated phospholipids are well recognized, A report that catal ytically active endonuclear CTP:choline-phosphate cytidylyltransferase cu i s necessary for cell survival questions whether endonuclear, CDP-choline pa thway phosphatidylcholine synthesis may occur in situ. We report that chrom atin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pa thway. First, membrane-free nuclei retain all three CDP-choline pathway enz ymes in proportions comparable with the content of chromatin-associated pho sphatidylcholine. Second, following supplementation of cells with deuterate d choline and using electrospray ionization mass spectrometry, both the tim e course and molecular species labeling pattern of newly synthesized endonu clear and whole cell phosphatidylcholine revealed the operation of spatiall y separate, compositionally distinct biosynthetic routes. Specifically, end ogenous and newly synthesized endonuclear phosphatidylcholine species are b oth characterized by a high degree of diacyl/alkylacyl chain saturation. Th is unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depleti on and when proliferation is restored, suggesting that endonuclear disatura ted phosphatidylcholine enrichment is essential and closely regulated. We p ropose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messenge rs. Furthermore, our estimates of saturated phosphatidylcholine nuclear vol ume occupancy of around 10% may imply a significant additional role in regu lating chromatin structure.