Antineoplastic effect of compounds C14 and P8 on TNBC and radioresistant TNBC cells by stabilizing the K-Ras4BG13D/PDE6δ complex.

Introduction: Breast cancer (BC) is the leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) representing one of the most aggressive and treatment-resistant subtypes. In this study, we aimed to evaluate the antitumor potential of C14 and P8 molecules in both TNBC and radioresistant TNBC cells. These compounds were chosen for their ability to stabilize the complex formed by the overactivated form of K-Ras4BG13D and its membrane transporter (PDE6δ). Methods: The antitumor potential of C14 and P8 was assessed using TNBC cell lines, MDA-MB-231, and the radioresistant derivative MDA-MB-231RR, both carrying the K-Ras4B> G13D mutation. We investigated the compounds' effects on K-Ras signaling pathways, cell viability, and tumor growth in vivo. Results: Western blotting analysis determined the negative impact of C14 and P8 on the activation of mutant K-Ras signaling pathways in MDA-MB-231 and MDA-MB-231RR cells. Proliferation assays demonstrated their efficacy as cytotoxic agents against K-RasG13D mutant cancer cells and in inducing apoptosis. Clonogenic assays proven their ability to inhibit TNBC and radioresistant TNBC cell clonogenicity. In In vivo studies, C14 and P8 inhibited tumor growth and reduced proliferation, angiogenesis, and cell cycle progression markers. Discussion: These findings suggest that C14 and P8 could serve as promising adjuvant treatments for TNBC, particularly for non-responders to standard therapies. By targeting overactivated K-Ras and its membrane transporter, these compounds offer potential therapeutic benefits against TNBC, including its radioresistant form. Further research and clinical trials are warranted to validate their efficacy and safety as novel TNBC treatments.

Circulating miRNAs as Noninvasive Biomarkers for PDAC Diagnosis and Prognosis in Mexico.

Among malignant neoplasms, pancreatic ductal adenocarcinoma (PDAC) has one of the highest fatality rates due to its late detection. Therefore, it is essential to discover a noninvasive, early, specific, and sensitive diagnostic method. MicroRNAs (miRNAs) are attractive biomarkers because they are accessible, highly specific, and sensitive. It is crucial to find miRNAs that could be used as possible biomarkers because PDAC is the eighth most common cause of cancer death in Mexico. With the help of microRNA microarrays, differentially expressed miRNAs (DEmiRNAs) were found in PDAC tissues. The presence of these DEmiRNAs in the plasma of Mexican patients with PDAC was determined using RT-qPCR. Receiver operating characteristic curve analysis was performed to determine the diagnostic capacity of these DEmiRNAs. Gene Expression Omnibus datasets (GEO) were employed to verify our results. The Prisma V8 statistical analysis program was used. Four DEmiRNAs in plasma from PDAC patients and microarray tissues were found. Serum samples from patients with PDAC were used to validate their overexpression in GEO databases. We discovered a new panel of the two miRNAs miR-222-3p and miR-221-3p that could be used to diagnose PDAC, and when miR-221-3p and miR-222-3p were overexpressed, survival rates decreased. Therefore, miR-222-3p and miR-221-3p might be employed as noninvasive indicators for the diagnosis and survival of PDAC in Mexican patients.

Synergistic effect of antagonists to KRas4B/PDE6 molecular complex in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.

Interplay Between the Histone Variant H2A.Z and the Epigenome in Pancreatic Cancer.

BACKGROUND: The oncogenic process is orchestrated by a complex network of chromatin remodeling elements that shape the cancer epigenome. Histone variant H2A.Z regulates DNA control elements such as promoters and enhancers in different types of cancer; however, the interplay between H2A.Z and the pancreatic cancer epigenome is unknown. OBJECTIVE: This study analyzed the role of H2A.Z in different DNA regulatory elements. METHODS: We performed Chromatin Immunoprecipitation Sequencing assays (ChiP-seq) with total H2A.Z and acetylated H2A.Z (acH2A.Z) antibodies and analyzed published data from ChIP-seq, RNA-seq, bromouridine labeling-UV and sequencing (BruUV-seq), Hi-C and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) in the pancreatic cancer cell line PANC-1. RESULTS: The results indicate that total H2A.Z facilitates the recruitment of RNA polymerase II and transcription factors at promoters and enhancers allowing the expression of pro-oncogenic genes. Interestingly, we demonstrated that H2A.Z is enriched in super-enhancers (SEs) contributing to the transcriptional activation of key genes implicated in tumor development. Importantly, we established that H2A.Z contributes to the three-dimensional (3D) genome organization of pancreatic cancer and that it is a component of the Topological Associated Domains (TADs) boundaries in PANC-1 and that total H2A.Z and acH2A.Z are associated with A and B compartments, respectively. CONCLUSIONS: H2A.Z participates in the biology and development of pancreatic cancer by generating a pro-oncogenic transcriptome through its posttranslational modifications, interactions with different partners, and regulatory elements, contributing to the oncogenic 3D genome organization. These data allow us to understand the molecular mechanisms that promote an oncogenic transcriptome in pancreatic cancer mediated by H2A.Z.

In Silico Identification and Characterization of circRNAs as Potential Virulence-Related miRNA/siRNA Sponges from Entamoeba histolytica and Encystment-Related circRNAs from Entamoeba invadens.

Ubiquitous eukaryotic non-coding circular RNAs regulate transcription and translation. We have reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica with esterified 3'ss and 5'ss. Their 5'ss GU-rich elements are essential for their biogenesis and their suggested role in transcription regulation. Here, we explored whether exonic, exonic-intronic, and intergenic circular RNAs are also part of the E. histolytica and E. invadens ncRNA RNAome and investigated their possible functions. Available RNA-Seq libraries were analyzed with the CIRI-full software in search of circular exonic RNAs (circRNAs). The robustness of the analyses was validated using synthetic decoy sequences with bona fide back splice junctions. Differentially expressed (DE) circRNAs, between the virulent HM1:IMSS and the nonvirulent Rahman E. histolytica strains, were identified, and their miRNA sponging potential was analyzed using the intaRNA software. Respectively, 188 and 605 reverse overlapped circRNAs from E. invadens and E. histolytica were identified. The sequence composition of the circRNAs was mostly exonic although different to human circRNAs in other attributes. 416 circRNAs from E. histolytica were virulent-specific and 267 were nonvirulent-specific. Out of the common circRNAs, 32 were DE between strains. Finally, we predicted that 8 of the DE circRNAs could function as sponges of the bioinformatically reported miRNAs in E. histolytica, whose functions are still unknown. Our results extend the E. histolytica RNAome and allow us to devise a hypothesis to test circRNAs/miRNAs/siRNAs interactions in determining the virulent/nonvirulent phenotypes and to explore other regulatory mechanisms during amoebic encystment.

131I-C19 Iodide Radioisotope and Synthetic I-C19 Compounds as K-Ras4B-PDE6δ Inhibitors: A Novel Approach against Colorectal Cancer-Biological Characterization, Biokinetics and Dosimetry.

In 40-50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B-PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B-PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and 131I-C19 compounds as inhibitors of the K-Ras4B-PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B-PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and 131I-C19 and the tumor radiation dose were also estimated. The K-Ras4B-PDE6δ stabilizer, 131I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that 131I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

p21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.

A class I histone deacetylase is implicated in the encystation of Entamoeba invadens.

Epigenetic mechanisms such as histone acetylation and deacetylation participate in regulation of the genes involved in encystation of Entamoeba invadens. However, the histones and target residues involved, and whether the acetylation and deacetylation of the histones leads to the regulation of gene expression associated with the encystation of this parasite, remain unknown. In this study, we found that E. invadens histone H4 is acetylated in both stages of the parasite and is more highly acetylated during the trophozoite stage than in the cyst. Histone hyperacetylation induced by Trichostatin A negatively affects the encystation of E. invadens, and this inhibition is associated with the downregulation of the expression of genes implicated in the synthesis of chitin, polyamines, gamma-aminobutyric acid pathways and cyst wall proteins, all of which are important in the formation of cysts. Finally, in silico analysis and activity assays suggest that a class I histone deacetylase (EiHDAC3) could be involved in control of the expression of a subset of genes that are important in several pathways during encystation. Therefore, the identification of enzymes that acetylate and/or deacetylate histones that control encystation in E. invadens could be a promising therapeutic target for preventing transmission of other amoebic parasites such as E. histolytica, the causative agent of amoebiasis in humans.

The activity of Aurora kinase B is required for dengue virus release.

Flaviviruses, such as Dengue (DENV), Zika, Yellow Fever, Japanese Encephalitis and West Nile are important pathogens with high morbidity and mortality. The last estimation indicates that ∼390 millions of people are infected by DENV per year. The DENV replicative cycle occurs mainly in the cytoplasm of the infected cells and different cytoplasmic, nuclear and mitochondrial proteins participate in viral replication. In this paper we analyzed the participation of Aurora kinase B (AurKB) in the DENV replicative cycle using the specific AurKB inhibitor ZM 447439. The kinase inhibition does not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells. Moreover, confocal microscopy analysis of DENV-infected ZM 447439-treated cells show a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release.

Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum.

Post-translational modifications of histone H3 N-terminal tails are key epigenetic regulators of virulence gene expression and sexual commitment in the human malaria parasite Plasmodium falciparum Here, we identify proteolytic clipping of the N-terminal tail of nucleosome-associated histone H3 at amino acid position 21 as a new chromatin modification. A cathepsin C-like proteolytic clipping activity is observed in nuclear parasite extracts. Notably, an ectopically expressed version of clipped histone H3, PfH3p-HA, is targeted to the nucleus and integrates into mononucleosomes. Furthermore, chromatin immunoprecipitation and next-generation sequencing analysis identified PfH3p-HA as being highly enriched in the upstream region of six genes that play a key role in DNA replication and repair: In these genes, PfH3p-HA demarcates a specific 1.5 kb chromatin island adjacent to the open reading frame. Our results indicate that, in P. falciparum, the process of histone clipping may precede chromatin integration hinting at preferential targeting of pre-assembled PfH3p-containing nucleosomes to specific genomic regions. The discovery of a protease-directed mode of chromatin organization in P. falciparum opens up new avenues to develop new anti-malarials.

KRas4B-PDE6δ complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer.

BACKGROUND: The GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer. METHODS: The crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded. RESULTS: According to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model. CONCLUSIONS: We identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

The small organic molecule C19 binds and strengthens the KRAS4b-PDEδ complex and inhibits growth of colorectal cancer cells in vitro and in vivo.

BACKGROUND: Colorectal cancer is the third most common cancer worldwide; and in 40% of all cases, KRAS4b-activating mutations occur. KRAS4b is transported by phosphodiesterase-6δ (PDEδ) to the plasma membrane, where it gets activated. PDEδ downregulation prevents redistribution and activation of KRAS4b. Thus, targeting the KRAS4b-PDEδ complex is a treatment strategy for colorectal cancer. METHODS: Using docking and molecular dynamics simulations coupled to molecular mechanics, the generalized born model and solvent accessibility (MMGBSA) approach to explore protein-ligand stability, we found that the compound ((2S)-N-(2,5-diclorofenil)-2-[(3,4-dimetoxifenil)metilamino]-propanamida), termed C19, bound and stabilized the KRAS4b-PDEδ complex. We investigated whether C19 decreases the viability and proliferation of colorectal cancer cells, in addition to knowing the type of cell death that it causes and if C19 decreases the activation of KRAS4b and their effectors. RESULTS: C19 showed high cytotoxicity in the colorectal cancer cell lines HCT116 and LoVo, with a stronger effect in KRAS-dependent LoVo cells. Importantly, C19 significantly decreased tumor size in a xenograft mouse model and showed lower side effects than 5-fluorouracil that is currently used as colorectal cancer treatment. CONCLUSIONS: Mechanistically, the cytotoxic effect was due to increased apoptosis of tumor cells and decreased phosphorylation of Erk and Akt. Therefore, our results suggest that C19 may serve as a promising new treatment for colorectal cancer.

PfAP2Tel, harbouring a non-canonical DNA-binding AP2 domain, binds to Plasmodium falciparum telomeres.

The telomeres of the malaria parasite Plasmodium falciparum are essential not only for chromosome end maintenance during blood stage development in humans but also to generate genetic diversity by facilitating homologous recombination of subtelomeric, multigene virulence families such as var and rifin. However, other than the telomerase PfTERT, proteins that act at P. falciparum telomeres are poorly characterised. To isolate components that bind to telomeres, we performed oligonucleotide pulldowns and electromobility shift assays with a telomeric DNA probe and identified a non-canonical member of the ApiAP2 family of transcription factors, PfAP2Tel (encoded by PF3D7_0622900), as a component of the P. falciparum telomere-binding protein complex. PfAP2Tel is expressed throughout the intra-erythrocytic life cycle and localises to the nuclear periphery, co-localising with telomeric clusters. Furthermore, EMSAs using the recombinant protein demonstrated direct binding of PfAP2Tel to telomeric repeats in vitro, while genome-wide chromatin immunoprecipitation followed by next generation sequencing corroborated the high specificity of this protein to telomeric ends of all 14 chromosomes in vivo. Taken together, our data describe a novel function for ApiAP2 proteins at chromosome ends and open new avenues to study the molecular machinery that regulates telomere function in P. falciparum.

Maf1 is a negative regulator of transcription in Trypanosoma brucei.

RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

Associations between whole peripheral blood fatty acids and DNA methylation in humans.

Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage.

Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (ß-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect ß-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-ß-tubulin antibody of E. histolytica. The anti-ß-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-ß-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of ß-tubulin is shown in the cytoplasm of E. histolytica trophozoites.

A new nucleocytoplasmic RhoGAP protein contributes to control the pathogenicity of Entamoeba histolytica by regulating EhRacC and EhRacD activity.

Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immunofluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.

Ribosomal RNA Genes in the Protozoan Parasite Leishmania major Possess a Nucleosomal Structure.

Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA genes in early-branched eukaryotes. Here we analyze the chromatin architecture and distribution of some histone modifications in the rRNA genes in the parasitic protozoon Leishmania major. Southern blots of MNase-partially-digested chromatin with DNA probes spanning the whole rRNA gene repeat showed that the intergenic spacer presents a tight nucleosomal structure, whereas the promoter region is practically devoid of nucleosomes. Intermediate levels of nucleosomes were found in the rRNA coding regions. ChIP assays allowed us to determine that H3K14ac, H3K23ac and H3K27ac, epigenetics marks that are generally associated with activation of transcription, are enriched in the promoter region. In contrast, H4K20me3, which is generally related to transcriptional silencing, was absent from the promoter region and intergenic spacer and enriched in the coding region. Interestingly, the distribution pattern for H3K9me3, generally linked to heterochromatin formation, was very similar to the distribution observed with the euchromatin marks, suggesting that this modification could be involved in transcriptional activation of rRNA genes in L. major.

Identification of repressive and active epigenetic marks and nuclear bodies in Entamoeba histolytica.

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.

Novel hypomorphic mutation in IKBKG impairs NEMO-ubiquitylation causing ectodermal dysplasia, immunodeficiency, incontinentia pigmenti, and immune thrombocytopenic purpura.

NF-κB essential modulator (NEMO) is a component of the IKK complex, which participates in the activation of the NF-κB pathway. Hypomorphic mutations in the IKBKG gene result in different forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males without affecting carrier females. Here, we describe a hypomorphic and missense mutation, designated c.916G>A (p.D306N), which affects our patient, his mother, and his sister. This mutation did not affect NEMO expression; however, an immunoprecipitation assay revealed reduced ubiquitylation upon CD40-stimulation in the patient's cells. Functional studies have demonstrated reduced phosphorylation and degradation of IκBα, affecting NF-κB recruitment into the nucleus. The patient presented with clinical features of ectodermal dysplasia, immunodeficiency, and immune thrombocytopenic purpura, the latter of which has not been previously reported in a patient with NEMO deficiency. His mother and sister displayed incontinentia pigmenti indicating that, in addition to amorphic mutations, hypomorphic mutations in NEMO can affect females.