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

Abstract

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hvl, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hvl protein and synthetic peptides from the amino and carboxyl domains of hvl. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro-and micronuclei confirmed the micronuclear acquisition of hvl early in conjugation, hvl staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hvl may be related to the elimination of hvl during the dynamic restructuring of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro-and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hvl functions in establishing a transcriptionally competent state of chromatin.

Histone H2A.Z Is Widely but Nonrandomly Distributed in Chromosomes of Drosophila melanogaster

“…H2A.Z is associated with transcriptionally active nuclei in both Tetrahymena and Drosophila (23,24), and recent genetic evidence suggests that H2A.Z regulates transcription in budding yeast. 3 The distribution of H2Av in Drosophila chromosomes appeared to correlate with transcriptional potential, in general, but not necessarily with specific sites of active transcription.…”

“…H2A.F/Z is essential in Tetrahymena, Drosophila, and mice, but its essential function is not known (20 -22). H2A.F/Z may play a role in transcriptional regulation since in Tetrahymena its expression is associated with the transcriptionally active macronucleus, and in Drosophila its incorporation into chromatin during development is coincident with the start of zygotic gene expression (23,24). The chromosome loss phenotype caused by mutation to the H2A.F/Z homolog in fission yeast, pht1, suggests that H2A.F/Z could also play a role, directly or indirectly, in chromosome segregation (25).…”

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

“…Proteins were electrophoresed on one-dimensional SDS gels or two-dimensional acid-urea followed by SDS gels and stained with Coomassie blue. (Stargell et al, 1993 Figure 2A). …”

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

“…Blots were then processed as described above for the anti-ubiquitin antiserum, except that a 1:20,000 dilution of the secondary antibody was used. The following histone antisera were used: anti-H2B (1:15,000 dilution), anti-H2A (1:20,000 dilution), anti-H4 (1:4,000 dilution) antisera (provided by Dr. C. David Allis, Rockefeller University, New York, NY) and anti-H2A.Z antiserum (1:10,000 dilution) (41).…”