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 G1. 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 HIl 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 (H 1M) containing 3-6 phosphates, and all histone H 3 molecules were phosphorylated. Exit of cells from anaphase correlated closely with the dephosphorylation of H3 to unphosphorylated H3 and with the dephosphorylation of H~M to subphosphorylated H1 containing 0-3 phosphates. Further dephosphorylation of subphosphorylated HI to unphosphorylated H1 occurred as these cells left telophase and entered G1. These experiments demonstrated that H1M 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 H I 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 HlM and H3 phosphorylations remove this restriction during mitosis.Many investigators have proposed that DNA activities might be controlled by modulating the structure of chromatin through reversible modifications of the histone proteins (for recent review see [l I). Experimental results from our laboratory have supported this concept by demonstrating that the modification of histones by phosphorylation is associated with changes in chromatin structure [2-101. The strongest evidence for a correlation between histone phosphorylation and chromatin structural change has been found at mitosis. For example, in synchronized cultured mammalian cells,...

al residues per tetramer go into an -helical conformation. Complexing inhibits the slow aggregation of ARE and GRK. Histones ARE and LAK also interact in a 1:1 molar ratio, but there is no increased a helicity upon complexing, and slow aggregation and /3-sheet formation occur. ARE and KAS also interact, but, upon complexing, there is no change in the tyrosine rigidity or in the circular dichroism. A pattern of the interactions between LAK, KAS, ARE, and GRK is presented. more weakly (D'Anna and Isenberg, 1974a). The complexes require salt for stability and, in fact, they were formed by adding salt to histone mixtures in water. One or both of the partner histones in the strong complexes, KAS-GRK and LAK-KAS, show marked conformational changes upon complexing, and the complex shows considerably more -helical content than the uncomplexed molecules.Skandrani et al. (1972) and Kelley (1973) have also reported a 1:1 complex of histones LAK and KAS, which was isolated by chromatographic fractionation of mixed his-4992 BIOCHEMISTRY, VOL.

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.

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