The Twists and Turns of Chromatin and Histone Modifications in Human Cells

Abstract

Quiescent cells exit the proliferative cell cycle from G1 phase and remain in a "resting" state until signals are given to leave quiescence and once again begin proliferation. The ability to enter and maintain quiescence is driven by many processes including the inhibition of key cell cycle factors at the transcription and protein levels. Changes also occur at the level of chromatin in quiescent cells at both local promoters and on a genome-wide scale. We have analyzed the chromatin changes between proliferating and quiescent fibroblasts and show that fibroblasts induced into quiescence via contact inhibition have increased chromatin compaction. We monitored the levels of ~50 histone modifications using quantitative mass spectrometry and found that most modifications were similar between proliferating and quiescent cells, but large changes existed on lysine 20 of histone 4. H4K20 fluctuated across the cell cycle where the unmodified form decreased and the monomethyl form increased after S phase. The di- and trimethyl forms are specific for quiescent cells and did not fluctuate during the cell cycle. We were unable to induce quiescence in proliferating fibroblasts by increasing H4K20me2 or H4K20me3 levels. However, higher H4K20me2 in proliferating cells caused an accumulation in G2. We found that knockdown of Suv4-20h1 and Suv4-20h2 in combination caused a decrease in chromatin compaction as measured by FISH, but knockdown of only Suv4-20h2, and not Suv4-20h1, led to an increased number of cells in S phase when fibroblasts were cultured as proliferating, or after 24 hrs of serum starvation to induce quiescence. This points to a role for H4K20me3 in slowing the passage through the cell cycle and during quiescence exit. Finally, we monitored the turnover of acetylation in proliferating and quiescent fibroblasts by adding labeled glucose and analyzing histones with mass spectrometry. We found that acetyl turnover in quiescent fibroblasts is reduced compared to proliferating fibroblasts, despite the steady-state levels being equal.