TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

Ubiquitination and degradation of MGMT by TRIM72 increases the sensitivity of uveal melanoma cells to Dacarbazine treatment

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults with high metastasis rates. The O6-methylguanine DNA methyl transferase (MGMT) is involved in chemoresistance of Dacarbazine (DTIC) treatment. Our previous study found that the combination of oncolytic adenovirus H101 and DTIC in the treatment of UM cells shows a synergistic antitumor effect mainly though down-regulation of MGMT. MGMT knockdown by shRNAs increases the sensitivity of uveal melanoma cells to DTIC treatment. The protein hemostasis of MGMT is important for the antitumor effect of DTIC. Tripartite motif-containing protein 72 (TRIM72) belongs to the tripartite motif (TRIM) proteins family and was identified as a novel E3 ligase for MGMT, which interacts with and mediates the ubiquitination of MGMT. TRIM72 knockdown increases the protein levels of MGMT, while reduces the ubiquitination of MGMT. Further study indicated that MGMT is highly expressed in UM cells, and the protein levels of MGMT and TRIM72 shows a negative correlation. UM cells that ectopically expressing TRIM72 shows increased sensitivity to DTIC treatment, which is consistent with the antitumor affect exhibited by H101. These results suggest that TRIM72 is a promising therapeutic target for UM treatment.

Behavioural consequences of Setd1a haploinsufficiency in mice: evidence for heightened emotional reactivity and impaired sensorimotor gating

ABSTRACTA number of studies implicate the loss of function (LoF) mutations affecting the histone methyl transferase SETD1A in the aetiology of a range of neurodevelopmental disorders including schizophrenia. Here, we examined the behavioural consequences of haploinsufficiency of Setd1a in a mouse model. We find evidence for changes in a number of phenotypes of relevance to schizophrenia, including increased anxiety-related behaviour, enhanced acoustic startle response, and decreased pre-pulse inhibition of acoustic startle. The sensorimotor gating deficits in Setd1a+/- mice could not be rescued by haloperidol or risperidone, suggesting that these antipsychotics are ineffective for ameliorating schizophrenia-relevant phenotypes in Setd1a+/- mice and point to deficits in neural systems other than the monoamine system. These phenotypes are emerging as key features of a number of other mouse models of rare neurodevelopmental disorders caused by LoF mutations in genes encoding epigenome modifiers suggesting they may act in a network to modulate brain development. Taken together these data strengthen the support for the use of Setd1a haploinsufficient mice as a model for the biological basis of schizophrenia, and point towards possible underpinning neural mechanisms.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Role of glycine-N-methyl transferase in human and murine nonalcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms of chronic liver disease worldwide, mirroring the obesity epidemic. NAFLD is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). This significantly increases the risk of cirrhosis and decompensated liver failure. Past studies in rodent models have shown that the knockout of glycine-N-methyltransferase (GNMT) is associated with steatosis, fibrosis, and hepatocellular carcinoma. However, the attenuation of GNMT in subjects with NASH and the molecular basis for its impact on the disease process have yet to be elucidated. To address this knowledge gap, we show the reduction of GNMT protein levels in the liver of NASH subjects compared to healthy controls. To gain insight into the impact of decreased GNMT in the disease process, we performed global label-free proteome studies on the livers from a murine Western diet-based model of NASH. We identified the differential expression of essential proteins involved in hallmark NASH pathogenesis, including lipid metabolism, inflammation, and fibrosis. Significantly, the downregulation of GNMT, the prominent regulator of S-adenosylmethionine (AdoMet), was identified as a contributing factor to these networks, increasing fourfold in AdoMet levels. AdoMet is an essential metabolite for transmethylation reactions and a substrate for polyamine synthesis, and its levels can impact polyamine flux and generate oxidative stress. Therefore, we performed targeted proteome and metabolomics studies to show a decrease in GNMT transmethylation, an increase in flux through the polyamine pathway, and increased oxidative stress.Abstract Figure

Isoliquiritigenin Alleviates Semen Strychni-Induced Neurotoxicity by Restoring the Metabolic Pathway of Neurotransmitters in Rats

Acute neurotoxicity of Semen Strychni can result in sudden death in epilepsy. The detoxification method and mechanism of Semen Strychni acute poisoning have not been clarified. This experiment focused on the mechanism of Semen Strychni neurotoxicity and the alleviation effects of isoliquiritigenin. The rats were intraperitoneally injected with Semen Strychni extract (125 mg/kg), followed by oral administration of isoliquiritigenin (50 mg/kg) for 7 days. FJ-B staining was used to evaluate the degree of injury on hippocampus neurons. The concentration of monoamines, amino acids, and choline neurotransmitters, the Dopamine (DA) and 5-hydroxytryptamine (5-HT) metabolic pathway in the hippocampus, cerebellum, striatum, prefrontal cortex, hypothalamus, serum, and plasma were detected by LC-MS/MS. The expression of neurotransmitter metabolic enzymes [catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO)] and neurotransmitter receptors [glutamate N-methyl-D-aspartic acid receptors (NMDARs) and gamma-aminobutyric acid type A receptor (GABRs)] were, respectively determined using ELISA and qRT-PCR. The results indicated that Semen Strychni induced neuronal degeneration in the hippocampal CA1 region. Meanwhile, Semen Strychni inhibited the mRNA expression of NMDAR1, NMDAR2A, NMDAR2B, GABRa1, GABRb2 and reduced the level of MAO, which disrupted the DA and 5-HT metabolic pathway. However, isoliquiritigenin reversed these effects. In summary, isoliquiritigenin showed alleviation effects on Semen Strychni-induced neurotoxicity, which could be attributed to restoring neurotransmitters metabolic pathway, most likely through the activation of NMDA receptors.