Immunofluorescence evidence for the absence of histone H1 in a mitotically dividing, genetically inactive nucleus.

Abstract

Antibodies directed against whole histone and purified lysine-rich histone H1extracted from isolated macronuclei of the ciliate Tetrahymena were obtained and conjugated to fluorescein isothiocyanate. The fluorescein-antibody conjugates were used to directly label Tetrahymena cells. Both macro-and micronuclei were visibly fluorescent in cells stained with anti-whole histone conjugate. However, the anti-H1 conjugate only labeled macronuclei. This in situ demonstration of the lack of positive immunofluorescent staining of micronuclei with anti-H 1 conjugate provides further evidence for the absence of H1 in the genetically inactive, mitotically dividing Tetrahymena micronucleus.The lysine-rich histone, H1, has been implicated in a variety of nuclear processes. Recent evidence links H1 and its phosphorylation in a number of cell types with the alteration of the structure of interphase chromatin throughout the cell cycle (16,18), including the conversion of nonproliferating cells to proliferating ones (17), rates of cell replication (1, 2, 28), chromosome condensation during mitosis (4,5,25), premitotic separation of sister chromatids (26), and the control of genetic activity (19,24,27).Studies done on the histones of the ciliate Tetrahymena have indicated that H1 is present, in multiple molecular species each capable of being phosphorylated, in the amitotically dividing, genetically active macronucleus of this organism (14). 1 However, the mitotically dividing, genetically inactive micronucleus apparently lacks histone H1 (12). These findings impose certain limits 1 Johmann, C. A., and M. A. Gorovsky. Manuscript in preparation.on the speculations concerning the biological role(s) of H1 in chromatin structure and function (12, 13).Since histone H1 is highly susceptible to proteolyric degradation (3, 30) and is easily dissociated from chromatin (10), the absence of H1 in isolated Tetrahymena micronuclei conceivably could be the result of a preparative artifact. Data obtained by Gorovsky and Keevert (12) argue strongly against such artifactual loss of H1 from micronuclei. However, studies done on isolated nuclei and chromatin cannot unequivocally dismiss the possibility that in vivo micronuclei contain H1. Thus, an immunofluorescence analysis was undertaken to determine the presence or absence of histone H1 in the macro-and micronuclei of Tetrahymena strain BVII. Antibodies directed against BVII whole histone and purified H1 were isolated, conjugated to fluorescein isothiocyanate, and used to stain BVII cells. The results confirm our previous conclusion that micronuclei lack histone H1 (12).

Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila.

“…Indirect immunofluorescence has already been utilized in several studies with starved Tetrahymena (3,9). However, the procedures used to "fix" the cells in these studies do not (at least in our hands) adequately maintain the integrity ofeither starved or conjugating cells (D. Wenkert and C. D. Allis, unpublished observations).…”

“…F I X A T 10 N O F C E L L S : All cells were stained by the indirect method. In preliminary experiments with conjugating cells, cells were fixed for immunomicroscopy as previously described (9) . However, these procedures led to considerable cell lysis especially with mating cells (see text).…”

Proteolytic processing of h1-like histones in chromatin: a physiologically and developmentally regulated event in Tetrahymena micronuclei.

“…Perhaps the most striking difference in histone composition between macro-and micronuclei of vegetative cells is seen with the linker-associated histones of the HI class (4,6). Macronuclei contain a typical H l which has been shown to be missing from micronuclei by biochemical (17) and immunofluorescent (22) analyses. Micronuclei, on the other hand, contain three unique polypeptides, a,/3, and % which do not have typical H1 solubility properties (C. D. Allis, R. L. Allen, and M. A. Gorovsky, unpublished observations), but which are associated with linker regions of micronuciear chromatin (4).…”

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

“…Macronuclei have an Hl-like protein that has many properties characteristic of H1 in higher organisms (Gorovsky et al 1974}. H1 is lacking in micronuclei (Gorovsky et al 1975;Johmann and Gorovsky 1976}. Instead, micronuclei contain a set of unique linker-associated polypeptides let, 13, "y, and 8) that lack typical Hl-like properties .…”