Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub.

Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.

Human chromosome-specific aneuploidy is influenced by DNA-dependent centromeric features.

Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.

Beyond the "First Hit": Marked Inhibition by N-Acetyl Cysteine of Chronic Ethanol Intake But Not of Early Ethanol Intake. Parallel Effects on Ethanol-Induced Saccharin Motivation.

BACKGROUND: A number of studies have shown that acetaldehyde synthesized in the brain is necessary to induce ethanol (EtOH) reinforcement in naïve animals (acquisition phase). However, after chronic intake is achieved (maintenance phase), EtOH intake becomes independent of acetaldehyde generation or its levels. Glutamate has been reported to be associated with the maintenance of chronic EtOH intake. The levels of brain extracellular glutamate are modulated by 2 glial processes: glutamate reabsorption via an Na(+) -glutamate transporter (GLT1) and a cystine-glutamate exchanger. Chronic EtOH intake lowers GLT1 levels and increases extracellular glutamate. The administration of N-acetyl cysteine (NAC), a precursor of cystine, has been shown to reduce the relapse of several drugs of abuse, while NAC has not been tested on chronic EtOH intake or on EtOH's influence on the motivation for another drug. These were investigated in the present study. METHODS: (i) Rats bred for their high EtOH intake were allowed access to 10% EtOH and water up to 87 days. NAC was administered (30 and 60 mg/kg daily, intraperitoneally) for 14 consecutive days, either during the acquisition phase or the maintenance phase of EtOH drinking. (ii) In additional experiments, rats were allowed EtOH (10%) and water access for 61 days, after which EtOH was replaced by saccharin (0.3%) to determine both if chronic EtOH consumption influences saccharin intake and whether NAC modifies the post chronic EtOH saccharin intake. RESULTS: NAC did not influence the acquisition ("first hit") of chronic EtOH intake, but greatly inhibited (60 to 70%; p < 0.0001) EtOH intake when NAC was administered to animals that were consuming EtOH chronically. NAC did not influence saccharin intake in naïve animals. In animals that had consumed EtOH chronically and were thereafter offered a saccharin solution (0.3%), saccharin intake increased over 100% versus that of EtOH-untreated animals, an effect that was fully suppressed by NAC. CONCLUSIONS: N-acetyl cysteine, a drug approved for use in humans, markedly reduces chronic EtOH intake and abolishes the increased intake of saccharin stimulated by chronic EtOH drinking.

First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxoscelesvenom agents.