Low levels of exogenous histone H1 in yeast cause cell death.

Abstract

To elucidate the hbnctin of lysine-rich histone, yeast cells, which are believed to lack this histone, were transformed with an expression vector carrying the sea urchin histone Hi gene under control of an inducible promoter. Expression of hill-length protein was tested by Immunobloffig and the intracellular distribution was monitored by immunoelectron microscopy. Even low amounts of exogenous H1 led to dramatic changes in ir lluar morphology and cell death. The cells that survived had lost either the plasmid or the ability to express the exogenous protein. Thus, even low amounts of canonical histone Hi are lethal to yeast cells.Histone H1 is known to bind to the linker DNA between nucleosomes, sealing off two turns of DNA around the histone octamer (1, 2). It is also involved in the formation and maintenance of higher-order chromatin structures and inhibits transcription by an as yet unknown mechanism (3, 4). One possible approach to elucidate the function of this histone would be to introduce exogenous H1 into appropriate living cells and follow its effects on the cellular function of interest. Yeast seems to be the obvious choice for such experiments, for it is believed not to contain an endogenous typical H1. Although immunological evidence for Hl-like proteins in yeast has been reported (5-7), such molecules might be quite different from the linker histones of higher eukaryotes. Moreover, the putative yeast H1 homologue is probably present in very low amounts and may be predominantly or exclusively localized in heterochromatic regions or at telomeres.Recently, we (8, 9) and others (10) Western Blotting. Total yeast proteins or 5% perchloric acid-extracted cellular proteins were fractionated electrophoretically in SDS-containing polyacrylamide gels (16) and transferred to nitrocellulose paper by semidry blotting in a model IMM-1 apparatus (W.E.P. Company, Seattle) as recommended by the manufacturer. Anti-mouse liver histone H1 antiserum (5) was presaturated with yeast lysate from the host strain by incubating nitrocellulose sheets (4 cm x 7 cm) with the lysate overnight at 40C and then with 10 ml ofanti-H1 antiserum for 3 hr at 370C. The filter with the adsorbed antibodies was removed and the remaining serum was used for Western blotting, performed according to routine procedures (5). Presaturation ofthe serum significantly reduced the background immunoreaction. Dilution of the anti-Hi antiserum was 1000-fold, and the enzyme substrate was o-phenylenediamine.Electron Microscopy. Cells were fixed for 5 min in 3.7% formaldehyde and then with 1% glutaraldehyde overnight at room temperature. Fixed cells were embedded in Lowacryl K4M (17) and ultrathin sections were examined in a JEOL JEM 100B electron microscope.Immune Microscopy. The immunostaining was performed directly on the microscopic grids at room temperature: ultrathin sections of Lowacryl-embedded fixed cells were incubated for 1 hr with presaturated serum (see above), which was diluted 400-fold in phosphate-buffered saline (PBS), washed in PBS,...

Expression of a Novel Leishmania Gene Encoding a Histone H1-Like Protein in Leishmania major Modulates Parasite Infectivity In Vitro

“…The mechanism by which the level of LNP18 expression affects Leishmania infectivity is not known yet. However, there is considerable evidence that histone H1 functions as a nonspecific repressor of transcription in higher eukaryotes and in Saccharomyces cerevisiae (36,61). Similar observations have been made for the protozoan Tetrahymena, which is evolutionarily close to Leishmania.…”

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

“…Multifocal chromatin condensation, however, was less evident in these pypl-expressing cells compared with Bax and Bak (our unpublished results). Similar morphological features have been described recently for S. cerevisiae overexpressing histone Hi (Miloshev et al, 1994), further suggesting that the morphology produced as a result of ectopic expression of Bax and Bak in S. pombe is not unique to these mammalian proapoptotic proteins.…”

Telomeric Position Effect Variegation in Saccharomyces cerevisiae by Caenorhabditis elegans Linker Histones Suggests a Mechanistic Connection between Germ Line and Telomeric Silencing

“…Human linker histone, which was abundantly expressed with the GAL10 promoter in budding yeast (3), did not enter the nuclear compartment and formed aggregates in the cytoplasm instead (W. Albig, personal communication). Expression of sea urchin linker histone in yeast caused cell death or greatly reduced the survival rate (29,31). In a control strain for which the URA3 In a sir3 mutant of the TPEV strain UCC3505 with abolished telomeric silencing, H1.1 expression did not affect URA3 expression.…”