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Micronuclei isolated from growing cells of Tetrahymena thermophila contain three H1-like polypeptides alpha, beta, and gamma. Micronuclei isolated from young conjugating cells (3-7 h) also contain a larger molecular weight polypeptide, X, which is being actively synthesized and deposited into these nuclei (Allis, C. D., and J. C. Wiggins, 1984, Dev. Biol., 101:282-294). Pulse-chase experiments (with growing and conjugating cells) suggested that X is a precursor to alpha and that alpha is further processed to gamma and a previously undescribed and relatively minor species, delta. These precursor-product relationships were supported by cross-reactivity with polyclonal antibodies raised against alpha and peptide mapping. While beta consistently became labeled under chase conditions (both in growing and mating cells), it was not clear whether it is part of the vivo processing event(s) which interrelates X, alpha, gamma, and delta. Beta was not recognized by alpha antibodies. Despite this uncertainty, these results suggest that proteolytic processing serves to generate significant changes in the complement of H1-like histones present in this nucleus.

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.

Histone H1 is highly phosphorylated in transcriptionally active, amitotic macronuclei of Tetrahymena during vegetative growth. However, the level of H1 phosphorylation changes dramatically in response to different physiological conditions. H1 is hyperphosphorylated in response to heat shock and during prezygotic stages of conjugation. Conversely, H1 is largely dephosphorylated during prolonged starvation and during elimination of parental macronuclei during conjugation. Mapping of phosphorylation sites within H1 indicates that phosphorylation occurs at multiple sites in the amino-terminal portion of the molecule, predominantly at threonine residues. Two of these sites have been identified by compositional analyses and microsequencing of tryptic peptides. Interestingly, two major sites contain the sequence Thr-Pro-Val-Lys similar to that contained in the sites recognized by growth-associated histone kinase in other organisms. No new sites are detected during the hyperphosphorylation of H1 which occurs during heat shock or in early stages of conjugation, and no sites are preferentially dephosphorylated during starvation or later stages of conjugation. Therefore, changes in the overall level of H1 phosphorylation, as opposed to phosphorylation or dephosphorylation at particular sites, appear to be important in the regulation of chromatin structure under these physiological conditions. Further, since no cell division or DNA replication occurs under these conditions, changes in the level of H1 phosphorylation are best correlated to changes in gene expression during heat shock, starvation, and conjugation. We suggest that, at least in Tetrahymena, H1 hyperphosphorylation is used as a rapid and transient mechanism for the cessation of transcription under conditions of cellular stress.

A salt-extracted histone acetyltransferase activity from Tetrahymena macronuclei acetylates mostly histone H3 and H4 when free histones are used as substrate. Free histone H4 is acetylated first at position 11 (monoacetylated) or positions 11 and 4 (diacetylated). This activity strongly resembles in vivo, deposition-related acetylation of newly synthesized histones. When acetylase-free mononucleosomes are used as substrate, all four core histones are acetylated by the same extract, and H4 is acetylated first at position 7 (monoacetylated) or positions 7 and 4 (diacetylated). In this respect, the activity of the extract is indistinguishable from postsynthetic, transcription-related histone acetylation that occurs in vivo or in isolated nuclei. Heat inactivation curves with both substrates are indistinguishable, and free histones compete with chromatin for limiting amounts of enzyme activity. These results argue strongly that two distinct, biologically important histone acetylations, one deposition related and one transcription related, are carried out by a single acetyltransferase.

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG-1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schröter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)