IN-VIVO PHOSPHORYLATION OF HISTONE H1 VARIANTS DURING THE CELL-CYCLE

In vivo phosphorylation of the five histone H1 variants H1a-H1e includ ing H1(0) in NIH 3T3 mouse fibroblasts was examined during the eel cyc le by using a combination of HPLC techniques and conventional AU gel e lectrophoresis. Phosphorylation starts during the late G1 phase and in creases throughout the S phase. In the late S phase, the H1 variants e xist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 ( H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphoryla ted protein ranging between 35% and 75%, according to the particular s ubtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphoryla tion maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosph orylated molecules. Phosphorylation of mouse H1c, however, occurs step wise during this transition. Phosphorylated mouse histone subtypes fro m cells in mitosis contain four phosphate groups in the case of H1a, H 1c, and H1e and five in the case of H1b and H1d. Comparison of the mou se phosphorylation pattern to that in rat C-6 glioma cells showed diff erences for H1e and H1d. By comparing the different phosphorylation pa tterns of the individual H1 variants during the cell cycle, we were ab le to classify the H1 histones into subtypes with low (H1a, H1c, H1(0) ) and high (H1b, H1d, H1e) phosphorylation levels.