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DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

JC polyomavirus (JCV, HPyV2) causes progressive multifocal leukoencephalopathy and has been linked to cancer development. JCV may naturally be highly prevalent in the human population and not just in diseased populations. The aim of this study was to the estimate the overall seroprevalence of JCV in the healthy human population by age of subjects, region or country of study, and assay methodology. A systematic review and meta-analysis with meta-regression using STATA 18. Pooled JCV seroprevalence from 25 population-level studies (N = 18,331) was 61% (95% CI, 56% - 66%, z = 33.84, p < 0.001). Meta-regression and subgroup analyses showed that subject age was the only predictive variable on JCV seroprevalence (z = 2.93, p = 0.003). Age was not normally distributed across seroprevalence. A detrended normal P-P plot under the cubic model with age on seroprevalence explained 99.9% of variance in the dataset (R2 = 0.999). The theories that grounded this study were the ecological systems theory, which support the study result that JCV infection appears ubiquitous in the human population and may be acquired early in life by two years of age. JCV seroprevalence starts high in early infant age, descends in late childhood/early adulthood, and starts to rise again towards older age, most probably, by viral reactivation due to immune senescence. The overall findings support the hypothesis that JCV is ubiquitous in the healthy human population, and not just diseased populations, which may have implications with JCV treatment, screening, and vaccine development. To the best of our knowledge, this is the first meta-analysis conducted on JCV seropositivity in healthy populations.

Since the advent of the all-volunteer force in the 1970s, marriage, parenthood, and family life have become commonplace in the U.S. military among enlisted personnel and officers alike, and military spouses and children now outnumber service members by a ratio of 1.4 to 1. Reviewing data from the government and from academic and nonacademic research, Molly Clever and David R. Segal find several trends that distinguish today's military families. Compared with civilians, for example, service members marry younger and start families earlier. Because of the requirements of their jobs, they move much more frequently than civilians do, and they are often separated from their families for months at a time. And despite steady increases since the 1970s in the percentage of women who serve, the armed forces are still overwhelmingly male, meaning that the majority of military parents are fathers. Despite these distinguishing trends, Celver and Segal's chief finding is that military families cannot be neatly pigeonholed. Instead, they are a strikingly diverse population with diverse needs. Within the military, demographic groups differ in important ways, and the service branches differ from one another as well. Military families themselves come in many forms, including not only the categories familiar from civilian life—two-parent, single-parent, and so on—but also, unique to the military, dual-service families in which both parents are service members. Moreover, military families' needs change over time as they move through personal and military transitions. Thus the best policies and programs to help military families and children are flexible and adaptable rather than rigidly structured.

In Arabidopsis , the RNA-directed DNA methylation pathway is important for establishing and maintaining DNA methylation — here Pol V is shown to depend on SUVH2 and SUVH9, where both of these proteins are proposed to bind specifically to methylated DNA to recruit Pol V, providing a self-reinforcing loop mechanism for maintenance of RNA-directed DNA methylation. Pol V recruitment to DNA methylation In the model plant Arabidopsis , the RNA-directed DNA methylation (RdDM) pathway is important for establishing and maintaining DNA methylation at genomic sites such as retrotransposons and heterochromatic repeats. The pathway involves two RNA polymerase complexes: Pol IV, which synthesizes small interfering RNAs, and Pol V, which generates non-coding transcripts that recruit downstream RdDM factors. Here, Steven Jacobsen and colleagues show that Pol V is dependent on two SRA/SET domain proteins, SUVH2 and SUVH9, for its activity and its recruitment to chromatin. SUVH2 and SUVH9 are proposed to bind specifically to methylated DNA in order to recruit Pol V, providing a self-reinforcing loop mechanism for maintenance of RdDM. This work suggests a mechanism for selectively targeting regions of plant genomes for epigenetic silencing. RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) 1 , 2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA 3 , 4 , 5 , 6 , 7 . The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown 8 , 9 . Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S -adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins 8 . SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA 8 , suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

UBE3A is an E3 ubiquitin ligase encoded by an imprinted gene whose maternal deletion or duplication leads to distinct neurodevelopment disorders Angelman and Dup15q syndromes. Despite the known genetic basis of disease, how changes in copy number of a ubiquitin ligase gene can have widespread impact in early brain development is poorly understood. Previous studies have identified a wide array of UBE3A functions, interaction partners, and ubiquitin targets, but no central pathway fully explains its critical role in neurodevelopment. Here, we review recent UBE3A studies that have begun to investigate mechanistic, cellular pathways and the genome-wide impacts of alterations in UBE3A expression levels to gain broader insight into how UBE3A affects the developing brain. These studies have revealed that UBE3A is a multifunctional protein with important nuclear and cytoplasmic regulatory functions that impact proteasome function, Wnt signaling, circadian rhythms, imprinted gene networks, and chromatin. Synaptic functions of UBE3A interact with light exposures and mTOR signaling and are most critical in GABAergic neurons. Understanding the genome-wide influences of UBE3A will help uncover its role in early brain development and ultimately lead to identification of key therapeutic targets for UBE3A-related neurodevelopmental disorders.

Artificial endonucleases consisting of a Fok I cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.

BackgroundLRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited.MethodsIn silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2’-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system.ResultsAcross breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2’-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer.ConclusionsOur study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.