Lozano-Amado Daniela, Herrera-Solorio Abril Marcela, Valdés Jesús, Alemán-Lazarini Leticia, Almaraz-Barrera Ma de Jesús, Luna-Rivera Eva, Vargas Miguel, Hernández-Rivas Rosaura
Molecular Biomedicine Department, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (IPN), Av. Instituto Politécnico Nacional # 2508, Apartado postal 14-740,, 07360, D. F. Mexico, México.
Biochemistry Department, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (IPN), Av. Instituto Politécnico Nacional # 2508, Apartado postal 14-740,, 07360, D. F. Mexico, México.
Parasit Vectors. 2016 Jan 14;9:19. doi: 10.1186/s13071-016-1298-7.
In human hosts, Entamoeba histolytica cysts can develop into trophozoites, suggesting that the life cycle of this parasite are regulated by changes in gene expression. To date, some evidence has suggested that epigenetic mechanisms such as DNA methylation and histone modification are involved in the regulation of gene expression in Entamoeba. Some post-translational modifications (PTMs) at the N-terminus of E. histolytica's histones have been reported experimentally, including tri-methylation in the lysine 4 of histone H3 (H3K4me3) and dimethylation in the lysine 27 of histone H3 (H3K27me2), dimethylation of arginine 3 (H4R3me2) and the indirect acetylation of histone H4 in the N-terminal region. However, it is not known which residues of histone H4 are subject to acetylation and/or methylation or where in the nucleus these epigenetic marks are located.
Histones from trophozoites of E. histolytica were obtained and analyzed by LC-MS/MS. WB assays were performed using antibodies against epigenetic marks (acetylated lysines and methylated arginines). Immunofluorescence assays (IFA) were carried out to determine the distribution of PTMs and the localization of DNA methylation as a heterochromatin marker. Nuclear bodies such as the nucleolus were identified by using antibodies against fibrillarin and nucleolin and speckles by using anti-PRP6 antibody.
Some new PTMs in histone H4 of E. histolytica, such as the acetylation of lysines 5, 8, 12 and 16 and the monomethylation of arginine 3, were identified by WB. IFA demonstrated that some marks are associated with transcriptional activity (such as acetylation and/or methylation) and that these marks are distributed throughout the E. histolytica nucleus. Staining with antibodies against anti-pan-acetylated lysine H4 histone and 5-methyl cytosine showed that the activation and transcriptional repression marks converge. Additionally, two nuclear bodies, the nucleolus and speckles, were identified in this parasite.
This study provides the first evidence that the nucleus of E. histolytica is not compartmentalized and contains two nuclear bodies, the nucleolus and speckles, the latter of which was not identified previously. The challenge is now to understand how these epigenetic marks and nuclear bodies work together to regulate gene expression in E. histolytica.
在人类宿主体内，溶组织内阿米巴包囊可发育为滋养体，这表明该寄生虫的生命周期受基因表达变化的调控。迄今为止，一些证据表明，DNA甲基化和组蛋白修饰等表观遗传机制参与了溶组织内阿米巴基因表达的调控。实验已报道了溶组织内阿米巴组蛋白N端的一些翻译后修饰（PTM），包括组蛋白H3赖氨酸4位点的三甲基化（H3K4me3）、组蛋白H3赖氨酸27位点的二甲基化（H3K27me2）、精氨酸3位点的二甲基化（H4R3me2）以及N端区域组蛋白H4的间接乙酰化。然而，尚不清楚组蛋白H4的哪些残基会发生乙酰化和/或甲基化，以及这些表观遗传标记在细胞核中的位置。
获取溶组织内阿米巴滋养体的组蛋白，并通过液相色谱-串联质谱（LC-MS/MS）进行分析。使用针对表观遗传标记（乙酰化赖氨酸和甲基化精氨酸）的抗体进行蛋白质印迹（WB）分析。进行免疫荧光分析（IFA）以确定PTM的分布以及作为异染色质标记的DNA甲基化的定位。通过使用抗纤维蛋白原抗体和抗核仁素抗体鉴定核仁等核体，使用抗PRP6抗体鉴定斑点。
通过WB鉴定出溶组织内阿米巴组蛋白H4中的一些新的PTM，如赖氨酸5、8、12和16位点的乙酰化以及精氨酸3位点的单甲基化。IFA表明一些标记与转录活性相关（如乙酰化和/或甲基化），并且这些标记分布在整个溶组织内阿米巴细胞核中。用抗全乙酰化赖氨酸H4组蛋白和5-甲基胞嘧啶抗体染色显示激活和转录抑制标记汇聚。此外，在这种寄生虫中鉴定出两个核体，即核仁和斑点。
本研究提供了首个证据，表明溶组织内阿米巴的细胞核没有分区，包含两个核体，即核仁和斑点，后者此前未被鉴定。现在面临的挑战是了解这些表观遗传标记和核体如何共同作用来调控溶组织内阿米巴的基因表达。
