Bacterial Outer Membrane Vesicles Induce a Transcriptional Shift in Arabidopsis Towards Immune System Activation Leading to Suppression of Pathogen Growth in Planta

Gram negative bacteria form spherical blebs on their cell periphery, which later dissociate and released into the surrounding environment. Previous studies have shown that these nano scale structures, derived primarily from the bacterial outer membrane and are termed outer membrane vesicles (OMVs), induce typical immune outputs in both mammals and plants. On the other hand, these same structures have been shown to promote infection and disease. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated samples at 2, 6 and 24 h post challenge. We found that the most pronounced transcriptional shift occurred in the first two time points, as was reflected by both the number of differentially expressed genes (DEGs) and the average fold change. Gene ontology enrichment analysis revealed that OMVs induce a major transcriptional shift in Arabidopsis towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors and transcriptional factors. Comparing Arabidopsis response to OMVs and to single purified elicitors, revealed that while OMVs induce a similar suite of genes and pathways as single elicitors, some differential pathways activated by OMVs were detected including response to drug and apoptosis, which may indicate exposure to toxic compounds via OMV. To examine whether the observed transcriptional shift in Arabidopsis leads to an effective immune response, plants were pretreated with OMVs and then inoculated with a bacterial pathogen. OMV-mediated priming led to a significant reduction in bacterial titer in inoculated leaves two days following inoculation. Mutations in the elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) receptor, did not significantly affect OMV-priming. All together these results show that OMV induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change is reflected in the ability to better resist bacterial infection.

A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer

Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.

Ilicicolin A Exerts Antitumor Effect in Castration-Resistant Prostate Cancer Via Suppressing EZH2 Signaling Pathway

The Polycomb protein enhancer of zeste homolog 2 (EZH2) has critical roles in prostate cancer (PCa) progression and drug-resistance, which remains an obstacle for PCa treatment. Enzalutamide (ENZ) is a second-generation androgen receptor antagonist employed for treatment of metastatic castration-resistant prostate cancer A considerable proportion of tumors eventually develop resistance during treatment. Thus, agents that can overcome resistance to PCa are needed urgently. Ilicicolin A (Ili-A), an ascochlorin derivative isolated from the coral-derived fungus Acremonium sclerotigenum GXIMD 02501, shows antiproliferative activity in human PCa cells, but its mechanism of action against Castration-resistant prostate cancer is not known. Herein, RNA-sequencing showed the EZH2 pathway to be involved in PCa proliferation. Ili-A at low doses reduced the protein level of EZH2, leading to transcriptional change. Interestingly, Ili-A suppressed the binding of EZH2 to promoter regions in AR/serine/threonine polo-like kinase-1/aurora kinase A. Moreover, Ili-A could enhance the anticancer activity of enzalutamide in CRPC cancer models. These data suggest that Ili-A could be used in combination with enzalutamide to treat CRPC.

Carbon Dioxide Pretreatment and Cold Storage Synergistically Delay Tomato Ripening through Transcriptional Change in Ethylene-Related Genes and Respiration-Related Metabolism

The effects of CO2 pretreatment before cold storage on tomato quality were investigated using physicochemical and transcriptome changes. Harvested tomatoes were treated with 30% or 60% CO2 for 3 h before storage at 4 °C for 14 d (cold storage), followed by transfer to 20 °C for 8 d (ambient conditions). The CO2-treated fruits were firmer with a better appearance than untreated fruits, even after being transferred from 4 °C storage to 20 °C for 8 d. CO2 pretreatment coupled with cold storage synergistically delayed tomato ripening by reducing respiration and lowering lycopene production. The tomatoes treated with 30% and 60% CO2 had fewer pits than untreated fruits after cold storage, even after being transferred to ambient conditions. Moreover, the 60% CO2 treatment significantly suppressed the decay rate. Transcriptome and metabolome functional enrichment analyses commonly showed the involvement of CO2-responsive genes or metabolites in sucrose and starch metabolism, as well as biosynthesis of secondary metabolites—in particular, glycolysis reduction. The most frequently detected domain was the ethylene-responsive factor. These results indicate that altered ethylene biosynthesis and ethylene signaling, via ethylene-responsive transcription factors and respiration-related pathways, appear to control CO2-induced fruit quality.

Photoperiod-dependent developmental reprogramming of the transcriptional response to seawater entry in Atlantic salmon (Salmo salar)

The developmental transition of juvenile salmon from a freshwater resident morph (parr) to a seawater (SW) migratory morph (smolt), known as smoltification, entails a reorganization of gill function to cope with the altered water environment. Recently, we used RNAseq to characterize the breadth of transcriptional change which takes place in the gill in the FW phase of smoltification. This highlighted the importance of extended exposure to short, winter-like photoperiods (SP) followed by a subsequent increase in photoperiod for completion of transcriptional reprogramming in FW and for efficient growth following transfer to SW. Here, we extend this analysis to examine the consequences of this photoperiodic history-dependent reprogramming for subsequent gill responses upon exposure to SW. We use RNAseq to analyse gill samples taken from fish raised on the photoperiod regimes we used previously and then challenged by SW exposure for 24-h. While fish held on constant light (LL) throughout were able to hypo-osmoregulate during a 24-h SW challenge, the associated gill transcriptional response was highly distinctive from that in fish which had experienced an 7 week period of exposure to SP followed by a return to LL (SPLL) and had consequently acquired the characteristics of fully developed smolts. Fish transferred from LL to SP, and then held on SP for the remainder of the study were unable to hypo-osmoregulate, and the associated gill transcriptional response to SW exposure featured many transcripts apparently regulated by the glucocorticoid stress axis and by the osmo-sensing transcription factor NFAT5. The importance of these pathways for the gill transcriptional response to SW exposure appears to diminish as a consequence of photoperiod mediated induction of the smolt phenotype, presumably reflecting preparatory developmetal changes taking place during this process.

Carbon Dioxide Pretreatment on Tomatoes Before Cold Storage Synergistically Delays Ripening Through Transcriptional Change of Ethylene-Related Genes and Respiration-Related Metabolisms

The effect of CO2 pre-treatments on tomato quality prior to cold storage was investigated using physiochemical and transcriptome changes. Three hours CO2 treated fruits were firmer than untreated fruits and had a good appearance even after being transferred from 4°C storage to 20°C for 8 d. CO2 pretreatment with cold storage showed a synergistic effect on delayed ripening through reduced respiration; these tomatoes exhibited a lower lycopene content than untreated fruit under cold storage. Tomatoes treated with 30% CO2 had fewer pits than untreated fruits subjected to chilling temperatures, even after being transferred to 20°C for 8 d. Functional enrichment analyses from transcriptome and metabolome commonly showed that CO2-responsive genes or metabolites were involved in the sucrose and starch and biosynthesis of secondary metabolisms. The most frequently detected domain, ethylene-responsive factor domain and reduced glycolysis provide insights into the mechanism that CO2 regulates tomato quality.

Blastocyst transfer in mice alters the placental transcriptome and growth

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.

Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a dual capability to self-renew and differentiate into all cell types necessary to develop an entire organism. Differentiation is associated with dynamic epigenetic alteration and transcriptional change, while self-renewal depends on maintaining the genome DNA accurately. Genome stability of PSCs is strictly regulated to maintain pluripotency. However, the DNA damage response (DDR) mechanism in PSCs is still unclear. There is accumulating evidence that genome stability and pluripotency are regulated by a transcriptional change in undifferentiated and differentiated states. iPSCs are ideal for analyzing transcriptional regulation during reprogramming and differentiation. This study aimed to elucidate the transcriptional alteration surrounding genome stability maintenance, including DNA repair, cell cycle checkpoints and apoptosis in fibroblasts, iPSCs and neural progenitor cells (NPCs) derived from iPSCs as differentiated cells. After ionizing radiation exposure, foci for the DNA double-stranded break marker γ-H2AX increased, peaking at 0.5 h in all cells (>90%), decreasing after 4 h in fibroblasts (32.3%) and NPCs (22.3%), but still remaining at 52.5% (NB1RGB C2 clone) and 54.7% (201B7 cells) in iPSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were detected, indicating that iPSCs’ apoptosis increases. In addition, RNA sequencing (RNA-Seq) analysis showed high expression of apoptosis genes (TP53, CASP3 and BID) in iPSCs. Results suggested that increased apoptosis activity maintains accurate, undifferentiated genome DNA in the cell population.