Distinct acetylation of Trypanosoma cruzi histone H4 during cell cycle, parasite differentiation, and after DNA damage

Abstract

Histones of trypanosomes are quite divergent when compared to histones of most eukaryotes. Nevertheless, the histone H4 of Trypanosoma cruzi, the protozoan that causes Chagas' disease, is acetylated in the N terminus at lysines 4, 10, and 14. Here, we investigated the cellular distribution of histone H4 containing each one of these posttranslational modifications by using specific antibodies. Histone H4 acetylated at lysine 4 (H4-K4ac) is found in the entire nuclear space preferentially at dense chromatin regions, excluding the nucleolus of replicating epimastigote forms of the parasite. In contrast, histone H4 acetylated either at K10 or K14 is found at dispersed foci all over the nuclei and at the interface between dense and nondense chromatin areas as observed by ultrastructural immunocytochemistry. The level of acetylation at K4 decreases in nonreplicating forms of the parasites when compared to K10 and K14 acetylations. Antibodies recognizing the K14 acetylation strongly labeled cells at G2 and M stages of the cell cycle. Besides that, hydroxyurea synchronized parasites show an increased acetylation at K4, K10, and K14 after S phase. Moreover, we do not observed specific colocalization of K4 modifications with the major sites of RNA polymerase II. Upon γ-irradiation that stops parasite replication until the DNA is repaired, dense chromatin disappears and K4 acetylation decreases, while K10 and K14 acetylation increase. These results indicate that each lysine acetylation has a different role in T. cruzi. While K4 acetylation occurs preferentially in proliferating situations and accumulates in packed chromatin, K10 and K14 acetylations have a particular distribution probably at the boundaries between packed and unpacked chromatin.

Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi

“…In this work, treatment with topo I inhibitors promoted severe unpacking of the heterochromatin in interphase cells, which is usually observed surrounding the nucleolus and juxtaposed to the inner nuclear membrane [7,54]. Such ultrastructural alterations were also observed in T. cruzi epimastigotes exposed to ␥ radiation [55]. Taken together, the effects of topo I inhibitors on growth impairment and nuclear structure alterations suggest that the T. cruzi cell cycle was arrested in order to repair the DNA.…”

Trypanosoma cruziDNA replication includes the sequential recruitment of pre-replication and replication machineries close to nuclear periphery

“…We, along with others, have already shown that it is possible to synchronize T. cruzi epimastigote cells with hydroxyurea. 9,17,18 Hydroxyurea inhibits the nucleotide reductase enzyme, thereby consuming dNTPs. In the absence of dNTPs, DNA elongation is blocked and most of the cells are arrested very early in S phase.…”

“…It has been shown that 18 h after the release, most of the cells are still at G 2 . 18 To examine cells just after mitosis, we took epimastigotes 20 h after hydroxyurea release. At this point, 25% of the cells were in cytokinesis (two nuclei, two kinetoplasts and two flagella) and 75% of cells had concluded the different patterns of tcorc1/cdc6 and tcpcna localization are related to the cell cycle.…”

Cell Biology of the Trypanosome Genome

“…They have been implicated in trypanosome transcription initiation and termination (154,162,174,191), cell cycle progression (70,85), and telomeric silencing (90). Acetylated histone H4 is distributed in a punctate pattern in the T. brucei and T. cruzi nucleoplasm (133,162,165). In the case of H4K10ac, this most likely reflects the association with pol II transcription start sites (162).…”