LncRNA Khps1 Regulates Expression of the Proto-oncogene SPHK1 via Triplex-Mediated Changes in Chromatin Structure.

Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis.

The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes.

Pairing orphan G protein­coupled receptors (GPCRs) with their cognate endogenous ligands is expected to have a major impact on our understanding of GPCR biology. It follows that the reproducibility of orphan receptor ligand pairs should be of fundamental importance to guide meaningful investigations into the pharmacology and function of individual receptors. GPR17 is an orphan receptor characterized by some as a dualistic uracil nucleotide/cysteinyl leukotriene receptor and by others as inactive toward these stimuli altogether. Whereas regulation of central nervous system myelination by GPR17 is well established, verification of activity of its putative endogenous ligands has proven elusive so far. Herein we report that uracil nucleotides and cysteinyl leukotrienes do not activate human, mouse, or rat GPR17 in various cellular backgrounds, including primary cells, using eight distinct functional assay platforms based on labelfree pathway-unbiased biosensor technologies, as well as canonical second-messenger or biochemical assays. Appraisal of GPR17 activity can neither be accomplished with co-application of both ligand classes, nor with exogenous transfection of partner receptors (nucleotide P2Y12, cysteinyl-leukotriene CysLT1) to reconstitute the elusive pharmacology. Moreover, our study does not support the inhibition of GPR17 by the marketed antiplatelet drugs cangrelor and ticagrelor, previously suggested to antagonize GPR17. Whereas our data do not disagree with a role of GPR17 per se as an orchestrator of central nervous system functions, they challenge the utility of the proposed (ant)agonists as tools to imply direct contribution of GPR17 in complex biologic settings.

TAF12 recruits Gadd45a and the nucleotide excision repair complex to the promoter of rRNA genes leading to active DNA demethylation.

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres.

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

Tracking climate-change-induced biological invasions by metabarcoding archived natural eDNA samplers.

In a time of unprecedented environmental change, understanding the response of organisms and ecosystems to change is paramount1. However, our knowledge of anthropogenic impacts on ecosystems is limited by a lack of standardized retrospective biomonitoring data2. Here, we use a four-decade time series of archived blue mussels to trace spatiotemporal biodiversity change in coastal ecosystems. The filter-feeding mussels, which were initially collected for pollution monitoring, can serve as natural eDNA samplers, carrying an imprint of the surrounding aquatic community at the time of sampling3. By sequencing the preserved DNA, we characterize highly diverse mussel-associated communities and reconstruct the invasion trajectory of an invasive species, the barnacle Austrominius modestus. We quantitatively trace population growth of the invader to the detriment of native taxa and uncover repeated population collapses and reinvasions after cold winters. By providing highly resolved temporal data on community assembly and global warming-driven invasion processes, natural eDNA sampler time series overcome a critical shortfall in our understanding of biodiversity change in the Anthropocene.

Lack of beta-arrestin signaling in the absence of active G proteins.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

Direct and Topoisomerase†II Mediated DNA Damage by Bis-3-chloropiperidines: The Importance of Being an Earnest G.

Bis-3-chloropiperidines are a new class of DNA-active compounds capable of alkylating nucleobases and inducing strand cleavage. In this study, we investigated the reactivity of these mustard-based agents with both single- and double-stranded DNA constructs. Polyacrylamide gel electrophoresis (PAGE) and electrospray ionization mass spectrometry (ESI-MS) were used to obtain valuable insight into their mechanism at the molecular level and to investigate their time- and concentration-dependent activity. The results revealed the preferential formation of mono- and bifunctional adducts at nucleophilic guanine sites. In a stepwise fashion, alkylation was followed by depurination and subsequent strand scission at the ensuing apurinic site. We demonstrated that the covalent modifications introduced by this new class of compounds can inhibit the activity of essential DNA-processing proteins, such as topoisomerase IIα, thereby suggesting that bis-3-chloropiperidines may have excellent anticancer potential.

Antiepileptic drugs affect neuronal androgen signaling via a cytochrome P450-dependent pathway.

Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

Gender-specific polygenic control of ethylnitrosourea-induced oncogenesis in the rat peripheral nervous system.

The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.