Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A-Treated Stem Cells

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications

Yb-1 oncoprotein controls pi3k/akt pathway by reducing pten protein level

YB-1 is a multifunctional protein overexpressed in many types of cancer. It is a crucial oncoprotein that regulates cancer cell progression and proliferation. Ubiquitously expressed in human cells, YB-1 protein functions are strictly dependent on its subcellular localization. In the cytoplasm, where YB-1 is primarily localized, it regulates mRNA translation and stability. However, in response to stress stimuli and activation of PI3K and RSK signaling, YB-1 moves to the nucleus acting as a prosurvival factor. YB-1 is reported to regulate many cellular signaling pathways in different types of malignancies. Furthermore, several observations also suggest that YB-1 is a sensor of oxidative stress and DNA damage. Here we show that YB-1 reduces PTEN intracellular levels thus leading to PI3K/Akt pathway activation. Remarkably, PTEN reduction mediated by YB-1 overexpression can be observed in human immortalized keratinocytes and HEK293T cells and cannot be reversed by proteasome inhibition. Real-time PCR data indicate that YB-1 silencing up-regulates the PTEN mRNA level. Collectively, these observations indicate that YB-1 negatively controls PTEN at the transcript level and its overexpression could confer survival and proliferative advantage to PTEN proficient cancer cells

Detection of malaria sporozoites expelled during mosquito sugar feeding.

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria

Identification of Cdk8 and Cdkn2d as New Prame-Target Genes in 2C-like Embryonic Stem Cells

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame’s downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes, Cdk8 and Cdkn2d, bound by Prame. These two targets, involved in cancer dedifferentiation and pluripotency, have been further validated in RA-resistant ESCs. Here, we observed for the first time that Prame controls the Cdk8 and Cdkn2d genes in ESCs after RA treatment, shedding light on the regulatory network behind the establishment of naïve pluripotency

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15\u201320 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

POZ-, AT-hook-, and Zinc Finger-containing Protein (PATZ) Interacts with Human Oncogene B Cell Lymphoma 6 (BCL6) and Is Required for Its Negative Autoregulation.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway

DNA Damage, Homology-Directed Repair, and DNA Methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments

Dysregulation of principal cell miRNAs facilitates epigenetic regulation of AQP2 and results in nephrogenic diabetes insipidus

Background MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression. Methods Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre1 mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice. Results The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre1 mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre1 mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre1 mice, resulting in decreased RNA Pol II association. Conclusions Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression

Helicobacter pylori regulates iNOS promoter by histone modifications in human gastric epithelial cells. [R. Pero* corresponding author]

Inducible nitric oxide synthase (iNOS) expression is altered in gastrointestinal diseases. Helicobacter pylori (Hp) infection may have a critical role in iNOS disregulation. We undertook this study to investigate possible chromatin changes occurring early during iNOS gene activation as a direct consequence of Hp???gastric cells interaction. We show that Hp infection is followed by different expression and chromatin modifications in gastric cells including (1) activation of iNOS gene expression, (2) chromatin changes at iNOS promoter including decreased H3K9 methylation and increased H3 acetylation and H3K4 methylation levels, (3) selective release of methyl-CpG-binding protein 2 from the iNOS promoter. Moreover, we show that Hp-induced activation of iNOS is delayed, but not eliminated, by the treatment with LSD1 inhibitors. Our data suggest a role for specific chromatin-based mechanisms in the control of human iNOS gene expression upon Hp exposure

Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu