Acetylation of histone H4K4 is cell cycle regulated and mediated by HAT3 in Trypanosoma brucei

Abstract

SummaryPost-translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a 'histone code' that specifies epigenetic information for transcription regulation. With a limited number of histone-modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone-modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0-specific masking of the site, owing to non-covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.

Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging

“…Tubes to collect the sorted cells were coated with bovine serum albumin, to prevent cells from sticking to the wall of the tubes. The sorted cells were fixed in suspension with formaldehyde and analyzed by fluorescence microscopy as described previously [4]. In agreement with published literature [3], G1 cells contained one nucleus and one kinetoplast and G2/M cells contained one nucleus and two kinetoplast ( Fig.…”

“…Similarly, an increase of temperature should be accompanied by a drop of RFI (0.001 per 2.5°C). To reduce the distance between coverslip and specimen, cells were not settled onto the slide but were settled directly onto aminopropyltriethoxysilane-coated coverslips, as described previously [4]. In addition, the attached cells were centrifuged at 800 × g for 2 min at room temperature, to achieve flat adhesion, as spherical aberration increases with specimen thickness.…”

“…To test this application, we imaged cells fluorescently stained with an antibody to the unmodified histone H4 lysine 4 site (H4K4unmod). This histone mark is strongly enriched during nuclear S phase [4]. If fluorescence emission is measured at different wavelengths, it is important to correct for chromatic aberration.…”

“…For example, cells with an elongated kinetoplast would be in the G2. However, our recent studies suggested that elongated kinetoplasts exist in nuclear S phase as well as [4].The goal of this study was to determine the cell-cycle stage of individual cells solely by their nDNA content. Using modern fluorescence microscopy and deconvolution algorithms, we * Corresponding author.…”

“…DNA of fixed or live cells was stained with propidium iodide or with DCO, respectively. Cells were sorted according to their total DNA content (this reflects nDNA content because kDNA represents only ~5% of total DNA in T. brucei [8]), then analyzed the sorted cells by fluorescence microscopy, as described previously [4]. Tubes to collect the sorted cells were coated with bovine serum albumin, to prevent cells from sticking to the wall of the tubes.…”