Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.XIST | X inactivation | MeCP2 | Rett syndrome | BMP/TGF-β

The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) plays a critical role in the maintenance of cellular redox and metabolic homeostasis, as well as the regulation of inflammation and cellular detoxication pathways. The contribution of the NRF2 pathway to organismal homeostasis is seen in many studies using cell lines and animal models, raising intense attention towards targeting its clinical promise. Over the last three decades, an expanding number of clinical studies have examined NRF2 inducers targeting an ever-widening range of diseases. Full understanding of the pharmacokinetic and pharmacodynamic properties of drug candidates rely partly on the identification, validation, and use of biomarkers to optimize clinical applications. This review focuses on results from clinical trials with four agents known to target NRF2 signaling in preclinical studies (dimethyl fumarate, bardoxolone methyl, oltipraz, and sulforaphane), and evaluates the successes and limitations of biomarkers focused on expression of NRF2 target genes and others, inflammation and oxidative stress biomarkers, carcinogen metabolism and adduct biomarkers in unavoidably exposed populations, and targeted and untargeted metabolomics. While no biomarkers excel at defining pharmacodynamic actions in this setting, it is clear that these four lead clinical compounds do touch the NRF2 pathway in humans.

BackgroundThe long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo.ResultsBy using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2–5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level.ConclusionOur results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0219-8) contains supplementary material, which is available to authorized users.

Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication timing, it remains unclear how transcription might impinge on replication, or vice versa . Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.