L.R. Gurley

Histone phosphorylation and chromatin structure were examined in synchronized CHO Chinese hamster cells during progression through mitosis. Cell population distribution in various phases of mitosis was determined by electron microscopy. Entry into mitosis was seen to occur in two stages: (1) the gathering of chromatin into aggregates of dense chromatin clumps during preprophase, followed by (2) the condensation of these aggregates into chromosome structures during prophase. Exit from mitosis was observed essentially as the reverse process, chromosomes first being disorganized into dense chromatin clumps during telophase, followed by dispersion of these aggregates in early G 1 . Correlating these structural changes with histone phosphorylation revealed that interphase‐type histone H1 phosphorylation (H1 I ) involving 1–3 phosphates per molecule existed in interphase and during the chromatin aggregation stages of mitosis (preprophase and telophase). Also, no histone H3 phosphorylation occurred during these periods of the cell cycle. It is proposed that H1 I phosphorylation may be involved with the submicroscopic changes in chromatin organization observed during interphase using molecular probes of chromatin structure. However, during mitosis, histone phosphorylation was correlated with microscopic chromatin structural changes. During the second stage of mitosis (prophase, metaphase, and anaphase), when chromosome structures were fully condensed, virtually all histone H1 existed as superphosphorylated molecules (H1 M ) containing 3–6 phosphates, and all histone H3 molecules were phosphorylated. Exit of cells from anaphase correlated closely with the dephosphorylation of H3 to unphosphorylated H3 and with the dephosphorylation of H1 M to subphosphorylated H1 containing 0–3 phosphates. Further dephosphorylation of subphosphorylated H1 I to unphosphorylated H1 occurred as these cells left telophase and entered G 1 . These experiments demonstrated that H1 M superphosphorylation and H3 phosphorylation are strictly mitotic events which occur only when chromosomes are fully condensed. The absence of Colcemid in some of these experiments eliminates the possibility that H1 M and H3 phosphorylations are artifacts of the Colcemid treatment. It is proposed that histones H1 and H3 may impose a restriction on chromatin structure which prevents chromosome condensation during interphase and that the H1 M and H3 phosphorylations remove this restriction during mitosis.

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G(1)-arrested and early G(1)-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G(2)/M boundary and traversing prophase. Since these phosphorylation events do not occur in G(1), S, or G(2) and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled (32)PO(4) from those specific histone fractions during transition of metaphase cells into interphase G(1) cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.

This paper presents the first unified quantitative study of the effects of butyrate concentration upon (1) cell-cycle progression, (2) modification of all inner histones, (3) dephosphorylation of histone H1, and (4) enhancement of an H1-like protein (BEP) in CHO cells. Flow cytometric analysis shows that exposure to butyrate enriches CHO cultures in G1 cells and, at sufficient butyrate concentration, leads to G1 arrest. Additionally, butyrate alters the rate of cell-cycle progression through G2/M and through S. Two-dimensional polyacrylamide electrophoresis and radiolabeling in butyrate-treated cultures indicate the presence of at least one site of internal acetylation in histone H2A, four sites of internal acetylation in histone H2B, five sites of internal acetylation in histone H3, and four sites of internal acetylation in histone H4. Histone H2A is also appreciably phosphorylated, so that it is acetylated and phosphorylated at a total of up to three sites. The distribution of modified species for all the inner (core) histones has been quantified from two-dimensional gels by using the three different methods of analysis? (1) direct densitometry of excised portions of the gel, (2) scintillation spectrometry of 3H-labeled histones, and (3) microdensitometry of photographic negatives. At 15 mM butyrate, 26% of H2B is acetylated at three to four sites, 37% of H3 is acetylated at three to five sites, and 50% of H4 is acetylated at three to four sites. Histone H1 is dephosphorylated as a function of butyrate concentration, and the dephosphorylation can be correlated with an increased proportion of G1 cells in culture. There is also a significant increase in the cellular content of two other proteins when cells are exposed to butyrate. The increase in one of these, BEP, has been quantified as a function of butyrate concentration after 24 h of exposure to butyrate. BEP appears to be related to histone H1O [Panyim, S., & Chalkley, R. (1969) Biochem. Biophys. Res. Commun. 37, 1042] and to induced protein IP25 [Keppel, F., Allet, B., & Eisen, H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 653]. The other protein (UP), which has a molecular weight of approximately 15 000, has not been identified. Butyrate induces a twofold increase in the cellular content of UP and a change in the distribution of UP molecular species.

The phosphorylation of different amino acids in distinct regions of fl histone was studied in highly synchronized Chinese hamster cell populations (line CHO). The purified, 32P-labeled fl histone was bisected into NH,-terminal and COOH-terminal fragments with N-bromosuccinimide. Tryptic phosphopeptides from these fragments were resolved using sequential high voltage electrophoretic steps on paper. No phosphorylation was observed in early G,-arrested cells. Interphase phosphorylation began in late G, in the COOH-terminal portion of the molecule on serine. This event continued throughout S phase and persisted into mitosis. However, in mitosis additional phosphorylation was observed in the Cell Cycle-specific Phosphorylation of Histone fl