Martin A. Gorovsky

Phosphorylation of Histone H3 Is Required for Proper Chromosome Condensation and Segregation

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

Phosphorylation of Linker Histone H1 Regulates Gene Expression In Vivo by Mimicking H1 Removal

Two Tetrahymena strains were created by gene replacement. One contained H1 with all phosphorylation sites mutated to alanine, preventing phosphorylation. The other had these sites changed to glutamic acid, mimicking the fully phosphorylated state. Global gene expression was not detectably changed in either strain. Instead, H1 phosphorylation activated or repressed specific genes in a manner that was remarkably similar to the effects of knocking out the gene encoding H1. These studies demonstrate a role for H1 phosphorylation in the regulation of transcription in vivo and suggest that it acts by mimicking the partial removal of H1.

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

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.

Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin

In Tetrahymena cells, phosphorylation of linker histone H1 regulates transcription of specific genes. Phosphorylation acts by creating a localized negative charge patch and phenocopies the loss of H1 from chromatin, suggesting that it affects transcription by regulating the dissociation of H1 from chromatin. To test this hypothesis, we used FRAP of GFP-tagged H1 to analyze the effects of mutations that either eliminate or mimic phosphorylation on the binding of H1 to chromatin both in vivo and in vitro. We demonstrate that phosphorylation can increase the rate of dissociation of H1 from chromatin, providing a mechanism by which it can affect H1 function in vivo. We also demonstrate a previously undescribed ATP-dependent process that has a global effect on the dynamic binding of linker histone to chromatin.

Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes.

The in vivo association of histone H1 with specific genes in Tetrahymena thermophila was studied by using a simplified cross-linking and immunoprecipitation technique. Four genes were analyzed whose activities vary in three different developmental states (logarithmic growth, starvation, and conjugation). Hybridization of the immunoprecipitated DNA to cloned probes showed an inverse correlation between the level of immunoprecipitation with H1 antiserum and transcriptional activity. This represents the first demonstration of an alteration in histone H1-DNA interaction associated with developmental changes in transcriptional activity.

A conserved histone variant enriched in nucleoli of mammalian cells.

An antibody has been raised to Tetrahymena histone variant hv1 that specifically stains the macronucleus, but not the micronucleus, of Tetrahymena. This antiserum also stains small punctate regions in nucleoli of several mammalian cell lines. These observations suggest that this histone variant has been highly conserved in evolution and may be associated with transcribed sequences.

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

The level of chromatin structure at which DNase I recognizes conformational differences between inert and activated genes has been investigated. Bulk and ribosomal DNAs of Tetrahymena pyriformis were differentially labeled in vivo with [14C]- and [3H]-thymidine, respectively, utilizing a defined starvation-refeeding protocol. The 3H-labeled ribosomal genes were shown to be preferentially digested by DNase I in isolated nuclei. Staphylococcal nuclease digested the ribosomal genes more slowly than bulk DNA, probably owing to the higher GC content of rDNA. DNase I and staphylococcal nuclease digestions of purified nucleosomes and of nucleosome core particles isolated from dual-labeled, starved-refed nuclei were indistinguishable from those of intact nuclei. We conclude from these studies that DNase I recognizes an alteration in the internal nucleosome core structure of activated ribosomal genes.

Conservation of intron position indicates separation of major and variant H2As is an early event in the evolution of eukaryotes.

SummaryGenomic clones ofDrosophila andTetrahymena histone H2A variants were isolated using the corresponding cDNA clones, (van Daal et al. 1988; White et al. 1988). The site corresponding to the initiation of transcription was defined by primer extension for bothDrosophila andTetrahymena genomic sequences. The sequences of the genomic clones revealed the presence of introns in each of the genes. TheDrosophila gene has three introns: one immediately following the initiation codon, one between amino acids 26 and 27 (gln and phe), and one between amino acids 64 and 65 (glu and val). TheTetrahymena gene has two introns, the positions of which are identical to the first two introns of theDrosophila gene. The chicken H2A.F variant gene has been recently sequenced and it contains four introns (Dalton et al. 1989). The first three of these are in the same positions as the introns in theDrosophila gene. The fourth intron interrupts amino acid 108 (gly). In all cases the sizes and the sequences of the introns are divergent. However, the fact that they are in conserved positions suggests that at least two of the introns were present in the ancestral gene. A phylogenetic tree constructed from the sequences of the variant and major cell cycle-regulated histone H2A proteins from several species indicates that the H2A variant proteins are evolutionarily separate and distinct from the major cell cycle-regulated histone H2A proteins. The ancestral H2A gene must have duplicated and diverged before fungi and ciliates diverged from the rest of the eukaryote lineage. In addition, it appears that the variant histone H2A proteins analyzed here are more conserved than the major histone H2A proteins.

Enzyme activity dot blots: a rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose.

Methods are described for assaying (Tetrahymena) histone acetyltransferase activity and (Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.

Histone H2B ubiquitylation is not required for histone H3 methylation at lysine 4 in tetrahymena.

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.