title: Identification of repressive and active epigenetic marks and nuclear bodies in Entamoeba histolytica.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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. dlamado@cinvestav.mx.
authors.forename: Daniela
authors.initials: D
authors.lastname: Lozano-Amado
authors.affiliation: 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. marvil_17@hotmail.com.
authors.forename: Abril Marcela
authors.initials: AM
authors.lastname: Herrera-Solorio
authors.affiliation: 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. jvaldes@cinvestav.mx.
authors.forename: Jesús
authors.initials: J
authors.lastname: Valdés
authors.affiliation: 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. letylazarinialeman@gmail.com.
authors.forename: Leticia
authors.initials: L
authors.lastname: Alemán-Lazarini
authors.affiliation: 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. madejesusalmab@yahoo.com.mx.
authors.forename: Ma de Jesús
authors.initials: Mde J
authors.lastname: Almaraz-Barrera
authors.affiliation: 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. eva_m_luna@yahoo.com.mx.
authors.forename: Eva
authors.initials: E
authors.lastname: Luna-Rivera
authors.affiliation: 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. mavargas@cinvestav.mx.
authors.forename: Miguel
authors.initials: M
authors.lastname: Vargas
authors.affiliation: 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. rohernan@cinvestav.mx.
authors.forename: Rosaura
authors.initials: R
authors.lastname: Hernández-Rivas
mesh_terms: D000107:Acetylation; D001120:Arginine; D019175:DNA Methylation; D004748:Entamoeba histolytica / Q000235:genetics*; D004749:Entamoebiasis / Q000382:microbiology*; D044127:Epigenesis, Genetic; D006657:Histones / Q000235:genetics; D006801:Humans; D008239:Lysine; D053719:Tandem Mass Spectrometry
chemical_list: D006657:Histones; D001120:Arginine; D008239:Lysine