Histone H3 phosphorylation of mammalian chromosomes

Inferences about the role and location of phosphorylated histone H3 are derived primarily from biochemical studies. A few direct observations at chromosome level have shown that phosphorylation begins at the site of heterochromatin and spreads throughout the chromosome. However, a comparative study of chromosomes of mouse (L929 cells), Chinese hamster (CHO 9 cells) and the Indian muntjac (male cells) reveals some distinguishable details among mammalian species. Whereas the L929 cells exhibit the typical pattern of phosphorylation at the region of centromeric heterochromatin associated with the active centromere, the heterochromatin blocks associated with the inactive centromeres also show label of about equivalent intensity. Throughout the cell cycle, heterochromatin exhibits sharper (denser) and better defined label than does euchromatin which expresses somewhat diffuse label. The centromere constriction on biarmed chromosomes, originating in Robertsonian translocations, appears phosphorylated in some, if not all chromosomes. A similar situation was found for the CHO 9 cells indicating that phosphorylation does include the region in which H3 is supposedly replaced by CENP-A. An interesting feature of the CHO cell line was the dense label at and near the telomeres; this feature was not observed in either the mouse or the Indian muntjac. The centromere regions of the Indian muntjac chromosomes showed three sites of label in the multicentric X chromosome and two each on chromosome pair number 1 and Y2; the sites coinciding with the reaction sites of antikinetochore antibodies. Also, the X and Y, chromosomes of Indian muntjac show intense phosphorylation at the sites of secondary constrictions. The chromosomes of all three species were phosphorylated throughout the cell cycle. As the chromosomes started to decondense during anaphase, heavy phosphorylation was observed in the form of discontinuous beaded structures indicating partial despiralization of the chromosome. Interestingly, when cells had completed karyokinesis and resolved into two independent nuclei, the phosphorylation was observed at the midbody. At this stage, the cytoplasm appeared to be again phosphorylated.