Modulation of linker histones during development in Tetrahymena: Selective elimination of linker histone during the differentiation of new macronuclei

doi:10.1016/0012-1606(85)90339-2

Developmental Biology

Volume 109, Issue 1, May 1985, Pages 1-8

https://doi.org/10.1016/0012-1606(85)90339-2 Get rights and content

Abstract

Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei. Instead, micronuclei contain three unique polypeptides, α, β, and γ, which are associated with linker regions of micronuclear chromatin. In this report polyclonal antibodies raised against macronuclear H1 are shown to react with α, β, and γ by immunoblotting analyses. This result suggests that these polypeptides share some common structural feature(s). Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence. However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen. These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena.

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Cited by (16)

Localization and functional analysis of HmgB3p, a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila
2013, Gene
Citation Excerpt :
The MLH (micronuclear linker histone, TTHERM_00471820) gene encodes a polyprotein comprising a set of four micronuclear linker histone proteins (alpha, beta, gamma, and delta) unrelated to Hho1p (macronuclear linker histone H1 protein) (Wu et al., 1994). Histone H1 and MLH proteins are chromatin proteins that associate with the inter-nucleosomal (linker) DNA (Allis et al., 1984; Chicoine et al., 1985). The micronuclear linker histones differ from each other, from macronuclear H1, and from the Hls of multicellular eukaryotes.
The high-mobility-group (HMG)-box domain represents a very versatile protein domain that mediates the DNA-binding of non-sequence-specific and sequence-specific proteins. HMG-box proteins are involved in various nuclear functions, including modulating chromatin structure and genomic stability. In this study, we identified the gene HMGB3 in Tetrahymena thermophila. The predicted HmgB3p contained a single HMG-box, an SK-rich-repeat domain and a neutral phosphorylated C-terminal. HMGB3 was expressed in the growth and starvation stages. Furthermore, HMGB3 showed a higher expression levels during the conjugation stage. HMGB3 knockout strains showed no obvious cytological defects, although initiation of HMGB3 knockout strain mating was delayed and maximum mating was decreased. HA-HmgB3p localized on the micronucleus (MIC) during the vegetative growth and starvation stages. Furthermore, HA-HmgB3p specially decorated the meiotic and mitotic functional MIC during the conjugation stage. Truncated HMGB3 lacking the HMG box domain disappeared from MICs and diffused in the cytoplasm. Overexpressed HmgB3p was abnormally maintained in newly developing macronuclei and affected the viability of progeny. Taken together, these results show that HmgB3p is a germline micronuclear-specific marker protein. It may bind to micronucleus-specific DNA sequences or structures and is likely to have some function specific to micronuclei of T. thermophila.
Linker histories are not essential and affect chromatin condensation in vivo
1995, Cell
We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila. These disruptions are shown to eliminate completely the expression of each protein. Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival. Histone H1 knockout (δH1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (δMicLH) cells have enlarged micronuclei and normal-sized macronuclei. δMicLH cells undergo mitosis normally. However, the micronuclear mitotic chromosome structure is less condensed. These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo.
A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions
1991, Analytical Biochemistry
Using the single cell eukaryote Tetrahymena thermophila, a simple method was developed for studying protein-DNA associations by cross-linking proteins to DNA with formaldehyde and immunoprecipitating the solubilized chromatin fragments with a specific antiserum. The protocol uses crude antiserum and involves only three steps: cross-linking, shearing to solubilize the chromatin, and immunoprecipitation. Methods for optimizing certain critical parameters, such as fixation time and NaCl concentration, are described. The method is likely to be generally useful for a variety of nuclear antigens.
Cell-cell interactions trigger the rapid induction of a specific high mobility group-like protein during early stages of conjugation in Tetrahymena
1991, Developmental Biology
Conjugation in Tetrahymena represents an ordered developmental pathway which represents the sexual phase of the ciliate life cycle. This pathway is initiated when starved cells of opposite mating types are mixed and are allowed to make a series of cell-cell contacts (a period termed costimulation) which lead to the formation of mating pairs. Here, we demonstrate that two previously described abundant high mobility group (HMG)-like proteins, HMG B and HMG C, whose synthesis appeared to be coordinately regulated in vegetative cells, are not required during the same stages of conjugation. The level of mRNA for both HMG B and HMG C is high during vegetative growth and during the development of new macronuclei. However, specific induction of HMG B mRNA is observed soon after cells of opposite mating types are mixed. Thus, the genes which encode HMG B and HMG C in Tetrahymena can be controlled independently or coordinately. Nuclear run-on experiments show that a significant factor underlying the rapid induction of HMG B message early in the sexual cycle is an increase in the transcriptional activity of the HMG B gene. Experiments are presented which show that this induction of HMG B message requires protein synthesis and is dependent upon the cell-cell contacts made during costimulation. Essentially all of the HMG B protein, which is newly synthesized during this period, is targeted to parental macronuclei where it serves an as yet undetermined function(s).
Unidirectional co-stimulation by a non-mating strain of Tetrahymena thermophila
1987, Experimental Cell Research
We report that a fatty acid auxotroph of Tetrahymena thermophila (RH179E1) fails to mate, yet retains the ability to co-stimulate normal cells unidirectionally. Thus, co-stimulation can be analyzed experimentally in the absence of pair formation. We show that the co-stimulation of normal cells of one mating type is sufficient to shorten the waiting period for pair formation of those cells with initiated cells. This is the first evidence that co-stimulation causes a hyperinduction of mating reactivity in T. thermophila, generating in turn a positive feedback mechanism for (presumably) gamone production. Co-stimulation by the variant strain is at a maximum after 3–4 h of exposure when the variant and wildtype cells are at a ratio of 1:1. When mixed with wild-type cells, RH179E1 induces the formation of progeny (at low frequency) which inherit exclusively genetic material of the wild-type cells.
Regulation of histone acetylation during macronuclear differentiation in Tetrahymena: Evidence for control at the level of acetylation and deacetylation
1986, Developmental Biology
During the postzygotic period of the sexual cycle (conjugation) in the ciliated protozoan, Tetrahymena, daughter products from a single micronuclear mitotic division develop into new macronuclei (anlagen) or new micronuclei depending upon their cytoplasmic location. In this study we have monitored the status of histone acetylation in synchronous populations of developing nuclei isolated from conjugating cells. Particular attention has been paid to the level of histone acetylation in new macronuclei following their differentiation from micronuclei. Like micronuclei isolated from vegetative cells (Vavra et al., 1982), micronuclei from conjugating cells (5 hr, 10–12 hr, and 15–16 hr) contain little if any acetylated histone and incorporate little postsynthetic acetate under any of our experimental conditions. In contrast, young new macronuclei (4C, 10–12 hr) incorporate significant amounts of acetate in vitro and in vivo provided that sodium butyrate is included during the labeling period. These results suggest that 4C anlagen contain both active acetylase and deacetylase activities even though the actual steady state level of acetylation found in these nuclei is low, more like that of micronuclei. At later stages of macronuclear maturation (8C, 15–16 hr), inner histones are hyperacetylated in a manner similar to parental, fully differentiated macronuclei. Furthermore, 8C anlagen incorporate acetate well even in the absence of sodium butyrate. Taken together these results suggest that endogenous deacetylase enzymes become either down-regulated and/or the rate of histone acetylases increases markedly during macronuclear differentiation.

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^☆: This work was supported by grants to C.D.A. from the American Cancer Society (CD-130) and the National Institutes of Health (HD 16259).

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Developmental Biology

Abstract

References (27)

Antibodies against the folding domain of histone H5 cross-react with H1° but not with H1

J. Biol. Chem

Identification and purification of young macronuclear anlagen from conjugating cells of Tetrahymena thermophila

Dev. Biol

Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila

Cell

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Dev. Biol

Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila

Exp. Cell Res

A conserved histone variant enriched in nucleoli of mammalian cells

Cell

Stable transcription complexes of Xenopus 5S genes: A means to maintain the differentiated state

Cell

The role of stable complexes that repress and activate eukaryotic genes

Cell

Isolation of micro- and macronuclei of Tetrahymena pyriformis

Conjugation in Tetrahymena thermophila: A temporal analysis of cytological stages

Exp. Cell Res

Serological detection of homologues of H1° with H5 and H1 histones

J. Biol. Chem

Affinity purification of antibodies from diazotized paper blots of heterogenous protein samples

J. Biol. Chem

The transcriptional regulation of Xenopus 5S RNA genes in chromatin: The roles of active stable transcription complexes and histone H1

Cell

Cited by (16)

Localization and functional analysis of HmgB3p, a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila

Linker histories are not essential and affect chromatin condensation in vivo

A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions

Cell-cell interactions trigger the rapid induction of a specific high mobility group-like protein during early stages of conjugation in Tetrahymena

Unidirectional co-stimulation by a non-mating strain of Tetrahymena thermophila

Regulation of histone acetylation during macronuclear differentiation in Tetrahymena: Evidence for control at the level of acetylation and deacetylation

Full paperModulation of linker histones during development in Tetrahymena: Selective elimination of linker histone during the differentiation of new macronuclei☆

Abstract

J. Biol. Chem

Dev. Biol

Cell

Dev. Biol

Exp. Cell Res

Cell

Cell

Cell

Exp. Cell Res

J. Biol. Chem

J. Biol. Chem

Cell

Full paper
Modulation of linker histones during development in Tetrahymena: Selective elimination of linker histone during the differentiation of new macronuclei☆