Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena

Abstract

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.

Figure 1

There is a single phosphorylation site, Ser-10, in the N terminus of Tetrahymena micronuclear H3. (A) HPLC-purified H3 from either macro- or micronuclei of logarithmically growing Tetrahymena cells labeled in vivo by [³²P]orthophosphate was resolved on a 12% SDS/PAGE gel and examined by Coomassie blue staining (Left) or autoradiography (Right). (B) In vivo phosphorylated micronuclear H3 was subjected to automated sequencing from the N terminus, and ³²P radioactivity eluted from each sequencing cycle was collected and counted. Note that an identical conclusion was reported previously by us as an unpublished observation (26).

Figure 2

The phosphorylated H3 antibody is specific for phosphorylated micronuclear H3^F. (A) Total proteins from either macro- (lane 1) or micronuclei (lane 2) of logarithmically growing Tetrahymena cells were resolved on a 12% SDS/PAGE gel, transferred to a nitrocellulose membrane, and probed by either phosphorylated H3 antibody (Left) or general H3 antibody (Right). (B) HPLC-purified H3 from micronuclei of logarithmically growing Tetrahymena cells was incubated in the presence (lane 2) or absence (lane 1) of alkaline phosphatase (AP) and subjected to immunoblotting by either phosphorylated H3 antibody (Upper) or general H3 antibody (Lower).

Figure 3

H3 phosphorylation is temporally correlated with mitosis in Tetrahymena. Logarithmically growing Tetrahymena cells were stained with the phosphorylated H3 antibody (B, D, F, H, and J) and the DNA-specific dye DAPI (A, C, E, G, and I). In A and B, a random population of cells is shown in low magnification. The closed arrows denote mitotic micronuclei; the open arrows denote interphase micronuclei, and the arrowheads denote macronuclei. The bar represents 10 μm. In C, D, E, F, G, H, I, and J, cells with micronuclei in interphase, mitosis, and S phase are shown in high magnification. The arrows denote micronuclei. The bar represents 10 μm.

Figure 4

H3 phosphorylation during Tetrahymena meiotic prophase. Early conjugating Tetrahymena cells were stained with the phosphorylated H3 antibody (B, D, F, H, and J) and DNA-specific dye DAPI (A, C, E, G, and I). The bar represents 10 μm. Numbered stages correspond to those described in ref. .

Figure 5

H3 phosphorylation is correlated with meiotic chromatin condensation in Tetrahymena. Late conjugating Tetrahymena cells were stained with the phosphorylated H3 antibody (B, D, F, and H) and DNA-specific dye DAPI (A, C, E, and G). The bar represents 10 μm.