Functional Analyses of <Emphasis Type="Italic">PtROS1</Emphasis>-RNAi in Poplars and Evaluation of Its Effect on DNA Methylation

DNA methylation occurs mostly at the C5 position of dinucleotide symmetric CpG sites in genomic DNA. A balance is maintained in the plant genome between DNA methylation mediated by RNA-directed DNA methylation (RdDM) and DNA demethylation mediated by the DEMETER (DME) protein family and REPRESSOR OF SILENCING (ROS1). We used double-stranded RNA (dsRNA) silencing to suppress ROS1 protein expression in ‘Nanlin895’ (Populus deltoides × Populus euramericana ‘Nanlin895’). Leaves of WT and transformant poplars revealed more symmetric methylation on CpG sites than roots and stems. In addition, leaves of transformant poplars revealed more methylated CpG sites in both 5.8S rDNA and histone H3 compared to WT types via 0, 50 and 100 mM NaCl treatments. In asymmetric methylation sites, transformant poplars exhibited more methylated CpHpG and CpHpH contexts than WT poplars. On the other hand, hypermethylation induced by PtROS1-RNAi construct resulted in pleiotropic phenotypic changes in transgenic poplars. The percentage of wavy leaves was increased maximum by ~45% in transgenic poplars. Also, the number of leaves was increased by ~200 number in transformants. Furthermore, shooting (%) and rooting (%) was decreased in transgenic poplars versus WT.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

Impact of <Emphasis Type="Italic">ZBTB7A</Emphasis> hypomethylation and expression patterns on treatment response to hydroxyurea

Background

We aimed to clarify the emerging epigenetic landscape in a group of genes classified as “modifier genes” of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.

Results

We examined the CpG islands’ DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients’ monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU.

Conclusions

This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5′ CpG sequences of all studied human HBB cluster “modifier genes.” Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the β-type hemoglobinopathy patients.

     

Epigenetic divergence of key genes associated with water temperature and salinity in a highly invasive model ascidian

Invasive species offer good models for studying mechanisms of response to rapid changing environments in the wild. DNA methylation is considered to be one of crucial drivers for rapid local adaptation. However, the extent of epigenetic variation and the factors driving such variation remain largely unexplored in biological invasions. Here we used direct bisulfite sequencing to investigate DNA methylation patterns of five key genes corresponding to two important environmental factors in marine ecosystems, water temperature and salinity, in a model invasive ascidian Ciona robusta (=C. intestinalis spA). Our results clearly showed that DNA methylation mainly occurred in gene bodies, rather than promoters, at regions with low values of CpG O/E. We detected significant variation of DNA methylation among populations in two genes (heat shock protein 90 and Na⁺-K⁺-2Cl^? cotransporter). Interestingly, significant correlation was detected between methylation levels and the two environmental factors at some CpGs in these two genes. When the data of all CpGs was subjected to principal component analysis, individuals were assigned back to their population orgins. All the results suggest that environmental factors likely contribute, at least partially, to the observed DNA methylation variation. Such variation, either by some loci alone or through gene networks, might be involved in rapid local adaptation during biological invasions.