Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

Molecular Markers For Selection of Resistance to Facial Eczema

<p>The disease facial eczema is caused by the fungal metabolite sporidesmin which produces photosensitisation of animals whose liver and biliary tract have been damaged by the toxin. Sporidesmin is produced by the pasture fungus Pithomyces chartarum and affects ruminant animals that graze on contaminated pasture. Previous studies have shown that sporidesmin is metabolised in the liver and have suggested that the toxin is metabolically inactivated by enzymes in the glutathione S-transferase and cytochrome P-450 families. The activities of these enzymes were therefore measured in liver extracts from Romneys that had been selected for resistance or susceptibility to sporidesmin - induced liver damage. Although there were no differences in cytochrome P-450 CO binding spectra or cytochrome c reductase between the selection lines, resistant Romneys had greater nitroanisole O-demethylase activity and this difference was apparently enhanced two days after dosing with sporidesmin. Dose-dependent differences occurred in the absence of major hepatocellular injury suggesting that they reflected changes in enzyme activity rather than changes in tissue mass. Aminopyrine N-demethylase did not vary significantly between the selection lines. Some differences in GSH-dependent metabolism were also observed. Undosed resistant Romneys showed greater GSH-dependent metabolism of sporidesmin in a spectrophotometric assay. It is possible that glutathione S-transferase Mu or Theta isoforms had greater activity in the resistant lines as differences were observed using p-nitrobenzyl chloride and 1,2 epoxy-3-p-nitrophenoxypropanol but not with 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene that are good substrates for these isoforms. 2-D PAGE was applied to the separation of whole homogenate and soluble proteins. Variations in expression of some proteins including GST Mu isoforms were found between the selection lines. Roles of cytochrome P-450 and glutathione S-transferase in the hepatic detoxication of sporidesmin have previously been demonstrated. Results obtained in this study suggest that resistant Romneys may have greater cytochrome P-450 O-demethylase and glutathione S-transferase activities that could be responsible for increased metabolic inactivation of sporidesmin. These differences may in the future be of use in design of DNA probes to enhance detection and selection of facial eczema resistant livestock.</p>

The Phytochemical Rhein Mediates M6A-Independent Suppression of Adipocyte Differentiation

Adipogenesis is mediated by the complex gene expression networks involving the posttranscriptional modifications. The natural compound rhein has been linked to the regulation of adipogenesis, but the underlying regulatory mechanisms remain elusive. Herein, we systematically analyzed the effects of rhein on adipogenesis at both the transcriptional and posttranscriptional levels. Rhein remarkably suppresses adipogenesis in the stage-specific and dose-dependent manners. Rhein has been identified to inhibit fat mass and obesity-associated (FTO) demethylase activity. Surprisingly, side-by-side comparison analysis revealed that the rhein treatment and Fto knockdown triggered the differential gene regulatory patterns, resulting in impaired adipocyte formation. Specifically, rhein treatment mildly altered the transcriptome with hundreds of genes dysregulated. N6-methyladenosine (m6A) methylome profile showed that, although the supply of rhein induced increased m6A levels on a small subset of messenger RNAs (mRNAs), few of them showed dramatic transcriptional response to this compound. Moreover, the specific rhein-responsive mRNAs, which are linked to mitotic pathway, are barely methylated or contain m6A peaks without dramatic response to rhein, suggesting separate regulation of global m6A pattern and adipogenesis mediated by rhein. Further identification of m6A-independent pathways revealed a positive regulator, receptor expressing-enhancing protein 3 (REEP3), in guidance of adipogenesis. Hence, this study provides the mechanistic view of the cellular actions of rhein in the modulation of adipogenesis and identifies a potential novel target for obesity therapeutic research.

SnRK1 stimulates the histone H3K27me3 demethylase JMJ705 to regulate a transcriptional switch to control energy homeostasis

Plant SNF1-Related Kinase1 (SnRK1) is an evolutionarily conserved energy-sensing protein kinase that orchestrates transcriptional networks to maintain cellular energy homeostasis when energy supplies become limited. However, the mechanism by which SnRK1 regulates this gene expression switch to gauge cellular energy status remains largely unclear. In this work, we show that the rice histone H3K27me3 demethylase JMJ705 is required for low energy stress tolerance in rice plants. The genetic inactivation of JMJ705 resulted in similar effects as those of the rice snrk1 mutant on the transcriptome, which impairs not only the promotion of the low energy stress-triggered transcriptional program but also the repression of the program under an energy-sufficient state. We show that the α-subunit of OsSnRK1 interacts with and phosphorylates JMJ705 to stimulate its H3K27me3 demethylase activity. Further analysis revealed that JMJ705 directly targets a set of low energy stress-responsive transcription factor genes. These results uncover the chromatin mechanism of SnRK1-regulated gene expression in both energy-sufficient and -limited states in plants and suggest that JMJ705 functions as an upstream regulator of the SnRK1α-controlled transcriptional network.

OS06.3A LSD1-directed therapy curtails glioblastoma tumorigenicity by limiting the adaptation of tumor initiating cells to stressful environments

BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

Identification of Jmjd3 as an Essential Epigenetic Regulator of Hox Gene Temporal Collinear Activation for Body Axial Patterning in Mice

Body axial patterning develops via a rostral-to-caudal sequence and relies on the temporal colinear activation of Hox genes. However, the underlying mechanism of Hox gene temporal colinear activation remains largely elusive. Here, with small-molecule inhibitors and conditional gene knockout mice, we identified Jmjd3, a subunit of TrxG, as an essential regulator of temporal colinear activation of Hox genes with its H3K27me3 demethylase activity. We demonstrated that Jmjd3 not only initiates but also maintains the temporal collinear expression of Hox genes. However, we detected no antagonistic roles between Jmjd3 and Ezh2, a core subunit of PcG repressive complex 2, during the processes of axial skeletal patterning. Our findings provide new insights into the regulation of Hox gene temporal collinear activation for body axial patterning in mice.

Growth arrest and DNA damage-inducible proteins (GADD45) in psoriasis

The interplay between T cells, dendritic cells and keratinocytes is crucial for the development and maintenance of inflammation in psoriasis. GADD45 proteins mediate DNA repair in different cells including keratinocytes. In the immune system, GADD45a and GADD45b regulate the function and activation of both T lymphocytes and dendritic cells and GADD45a links DNA repair and epigenetic regulation through its demethylase activity. Here, we analyzed the expression of GADD45a and GADD45b in the skin, dendritic cells and circulating T cells in a cohort of psoriasis patients and their regulation by inflammatory signals. Thirty patients (17 male/13 female) with plaque psoriasis and 15 controls subjects (7 male/8 female), were enrolled. Psoriasis patients exhibited a lower expression of GADD45a at the epidermis but a higher expression in dermal infiltrating T cells in lesional skin. The expression of GADD45a and GADD45b was also higher in peripheral T cells from psoriasis patients, although no differences were observed in p38 activation. The expression and methylation state of the GADD45a target UCHL1 were evaluated, revealing a hypermethylation of its promoter in lesional skin compared to controls. Furthermore, reduced levels of GADD45a correlated with a lower expression UCHL1 in lesional skin. We propose that the demethylase function of GADD45a may account for its pleiotropic effects, and the complex and heterogeneous pattern of expression observed in psoriatic disease.

Kdm3b haploinsufficiency impairs the consolidation of cerebellum-dependent motor memory in mice

Histone modifications are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifications in cerebellum-dependent motor learning and memory are not well understood. To test whether changes in histone methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+/−) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di-methylation is significantly increased selectively in the granule cell layer of the cerebellum of Kdm3b+/− mice. In the cerebellum-dependent optokinetic response (OKR) learning, Kdm3b+/− mice show deficits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA-seq analyses revealed that the expression levels of several plasticity-related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory.

Lysine Demethylases: Promising Drug Targets in Melanoma and Other Cancers

Epigenetic dysregulation has been implicated in a variety of pathological processes including carcinogenesis. A major group of enzymes that influence epigenetic modifications are lysine demethylases (KDMs) also known as “erasers” which remove methyl groups on lysine (K) amino acids of histones. Numerous studies have implicated aberrant lysine demethylase activity in a variety of cancers, including melanoma. This review will focus on the structure, classification and functions of KDMs in normal biology and the current knowledge of how KDMs are deregulated in cancer pathogenesis, emphasizing our interest in melanoma. We highlight the current knowledge gaps of KDMs in melanoma pathobiology and describe opportunities to increases our understanding of their importance in this disease. We summarize the progress of several pre-clinical compounds that inhibit KDMs and represent promising candidates for further investigation in oncology.