Histone Phosphorylation and Chromatin Structure during Mitosis in Chinese Hamster Cells

Abstract

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,...

ADP‐Ribosylation in Permeable HeLa S3 Cells

“…In the case of histones from metaphase cells there does not seem to be ADP-ribosylation of any large fraction of the total histone population. This contrasts with other known modification in mitosis such as phosphorylation of histone HI and substantial part of histone H3 [33].…”

N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation

“…Thus, disruption of N-myc results in a nearly complete loss of S-phase NPCs at E13 in several distinct germinal regions of the brain. To further examine the effects of loss of N-myc on the cell cycle, sections were stained with an antibody against phosphorylated histone H3 (phosphoH3), a marker for mitotic cells (Gurley et al 1978). Consistent with a substantial reduction in mitotic figures evident by H&E and DAPI staining (Fig.…”

Immunodetection of phosphohistone H3 as a surrogate of mitotic figure count and clinical outcome in cutaneous melanoma

“…It is typically no longer detectable when mitosis is completed. [6][7][8] Furthermore, phosphohistone H3 is a specific marker of mitotic figures, as it is not expressed in apoptotic bodies-common morphologic mimics of mitotic figures in routine light microscopy.…”