Placental endocrine insufficiency programs anxiety, deficits in cognition and atypical social behaviour in offspring.

Abnormally elevated expression of the imprinted PHLDA2 gene has been reported in the placenta of human babies that are growth restricted in utero in several studies. We previously modelled this gene alteration in mice and found that just 2-fold increased expression of Phlda2 resulted in placental endocrine insufficiency. In addition, elevated Phlda2 was found to drive fetal growth restriction (FGR) of transgenic offspring and impaired maternal care by their wildtype mothers. Being born small and being exposed to suboptimal maternal care have both been associated with the increased risk of mental health disorders in human populations. In the current study we probed behavioural consequences of elevated Phlda2 for the offspring. We discovered increased anxiety-like behaviours, deficits in cognition and atypical social behaviours, with the greatest impact on male offspring. Subsequent analysis revealed alterations in the transcriptome of the adult offspring hippocampus, hypothalamus and amygdala, regions consistent with these behavioural observations. The inclusion of a group of fully wildtype controls raised in a normal maternal environment allowed us to attribute behavioural and molecular alterations to the adverse maternal environment induced by placental endocrine insufficiency rather than the specific gene change of elevated Phlda2. Our work demonstrates that a highly common alteration reported in human FGR is associated with negative behavioural outcomes later in life. Importantly, we also establish the experimental paradigm that placental endocrine insufficiency can program atypical behaviour in offspring highlighting the under-appreciated role of placental endocrine insufficiency in driving disorders of later life behaviour.

Head-to-head antisense transcription and R-loop formation promotes transcriptional activation.

The mechanisms used by antisense transcripts to regulate their corresponding sense mRNAs are not fully understood. Herein, we have addressed this issue for the vimentin (VIM) gene, a member of the intermediate filament family involved in cell and tissue integrity that is deregulated in different types of cancer. VIM mRNA levels are positively correlated with the expression of a previously uncharacterized head-to-head antisense transcript, both transcripts being silenced in colon primary tumors concomitant with promoter hypermethylation. Furthermore, antisense transcription promotes formation of an R-loop structure that can be disfavored in vitro and in vivo by ribonuclease H1 overexpression, resulting in VIM down-regulation. Antisense knockdown and R-loop destabilization both result in chromatin compaction around the VIM promoter and a reduction in the binding of transcriptional activators of the NF-κB pathway. These results are the first examples to our knowledge of R-loop-mediated enhancement of gene expression involving head-to-head antisense transcription at a cancer-related locus.

A lncRNA Decoy Predicts Sensitivity to Cisplatin.

In a recent iScience paper by Fan et al., the long noncoding (lnc)RNA CISAL is shown to form a DNA-RNA triplex and to directly regulate BRCA1 transcription, thereby increasing cisplatin sensitivity and serving as a treatment efficacy biomarker. This opens promising avenues of research from both mechanistic and translational perspectives.

Unified Behavioral Scoring for Preclinical Models.

Preclinical mental health research relies upon animal models, and whilst many encouraging advances are being made, reproducibility and translational relevance may be limited by sub-optimal testing or model choices. Animal behaviors are complex and test batteries should be designed to include their multifaceted nature. However, multiple behavioral testing is often avoided due to cost, availability or statistical rigor. Additionally, despite the disparity in the incidence of mental health problems between the sexes, a move toward reducing animal numbers could be a deterrent to including both male and female animals. The current study introduces a unified scoring system for specific behavioral traits with the aim of maximizing the use of all data generated whilst reducing the incidence of statistical errors. Female and male mice from two common background strains were tested on behavior batteries designed to probe multiple aspects of anxiety-related and social behavioral traits. Results for every outcome measure were normalized to generate scores for each test and combined to give each mouse a single unified score for each behavioral trait. The unified behavioral scores revealed clear differences in the anxiety and stress-related, and sociability traits of mice. Principle component analysis of data demonstrated significant clustering of animals into their experimental groups. In contrast, individual tests returned an ambiguous mixture of non-significant trends and significant effects for various outcome measures. Utilizing a range of behavioral measures and combining all outcome measure data to produce unified scores provides a useful tool for detecting subtle behavioral traits in preclinical models.

The transcribed pseudogene RPSAP52 enhances the oncofetal HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent let-7 inhibition.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52. RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

Peg3 Deficiency Results in Sexually Dimorphic Losses and Gains in the Normal Repertoire of Placental Hormones.

Hormones from the fetally derived placenta signal to the mother throughout pregnancy to ensure optimal fetal growth and prepare the mother for her new role in nurturing her offspring. Through evolution, placental hormones have under gone remarkable diversification and species-specific expansions thought to be due to constant rebalancing of resource allocation between mother and offspring. Genomic imprinting, an epigenetic process in which parental germlines silence genes in the offspring, is thought to be the physical embodiment of a second conflicting interest, between the male and female mammal. Several genes silenced by paternal imprints normally function to limit the placental endocrine lineages of the mouse placenta. We hypothesized that paternal imprinting has adapted to overcome the rapid evolution of placental hormone gene families by directly regulating the lineages that express these hormones rather than individual hormones. This predicts the existence of genes maternally silenced in the offspring counteracting the influence of the paternal imprint. Here we report on the consequences of loss of function of Paternally expressed gene 3 (Peg3), on placental endocrine lineages. Mutant male placenta displayed a marked loss of the spongiotrophoblast, a key endocrine lineage of the placenta, and the glycogen cell lineage alongside reduced stores of placental glycogen and changes in expression of the normal repertoire of placental hormones. Peg3 is known to transcriptionally repress placental hormone genes. Peg3 consequently both positively and negatively regulates placental hormones through two independent and opposing mechanisms. Female placenta showed moderate response to loss of Peg3 with minor alterations to the junctional zone lineages and few changes in gene expression. These data highlight the important fact that female placenta compensate for the loss of Peg3 better than male placenta. This work lends further support to our novel hypothesis that the parental genomes are competing over the endocrine function of the mouse placenta and further suggests that a conflict between males and females begins in utero.

RNA-FISH to Study Regulatory RNA at the Site of Transcription.

The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris®, in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives. We have applied this method to the detection of nascent RNAs that partake of special regulatory structures called R loops. Their growing role in active gene expression regulation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012; Ginno et al., Mol Cell 45:814-825, 2012; Sun et al., Science 340:619-621, 2013; Bhatia et al., Nature 511:362-365, 2014) imposes the use of a combination of in vivo and in vitro techniques for the detailed analysis of the transcripts involved. Therefore, their study is a good example to illustrate how RNA FISH, combined with transcriptional arrest and/or cell synchronization, permits localization and temporal characterization of potentially regulatory RNA sequences.

Detection and Characterization of R Loop Structures.

R loops are special three stranded nucleic acid structures that comprise a nascent RNA hybridized with the DNA template strand, leaving a non-template DNA single-stranded. More specifically, R loops form in vivo as G-rich RNA transcripts invade the DNA duplex and anneal to the template strand to generate an RNA:DNA hybrid, leaving the non-template, G-rich DNA strand in a largely single-stranded conformation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012).DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation. RNA-DNA hybrids influence genomic instability, and growing evidence points to an important role for R loops in active gene expression regulation (Ginno et al., Mol Cell 45, 814-825, 2012; Sun et al., Science 340: 619-621, 2013; Bhatia et al., Nature 511, 362-365, 2014). Analysis of the occurrence of such structures is therefore of increasing relevance and herein we describe methods for the in vivo and in vitro identification and characterization of R loops in mammalian systems.R loops (DNA:RNA hybrids and the associated single-stranded DNA) have been traditionally associated with threats to genome integrity, making some regions of the genome more prone to DNA-damaging and mutagenic agents. Initially considered to be rare byproducts of transcription, over the last decade accumulating evidence has pointed to a new view in which R loops form more frequently than previously thought. The R loop field has become an increasingly expanded area of research, placing these structures as a major threat to genome stability but also as potential regulators of gene expression. Special interest has arisen as they have also been linked to a variety of diseases, including neurological disorders and cancer, positioning them as potential therapeutic targets [5].

Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer.

BACKGROUND: One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. RESULTS: Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. CONCLUSIONS: We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes.