Masahiro Uesaka

Bidirectional promoters are the major source of gene activation-associated non-coding RNAs in mammals

BackgroundThe majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples.ResultsWe found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs.ConclusionsWe propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.

Gene activation-associated long noncoding RNAs function in mouse preimplantation development

In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development. Highlighted article: During zygotic gene activation in the early mouse embryo, promoter-associated noncoding RNAs can promote expression of their partner mRNAs, which may be important for normal development.

MiR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Single-stranded noncoding RNAs mediate local epigenetic alterations at gene promoters in rat cell lines.

Background: Noncoding RNAs (ncRNAs) can alter epigenetic processes, mostly causing gene repression. Results: Antisense promoter-associated ncRNAs (pancRNAs) were associated with active chromatin marks at Nefl and Vim promoters. Forced expression and knockdown of these pancRNAs caused DNA demethylation and methylation, respectively. Conclusion: pancRNAs act in cis as a single-stranded form. Significance: Orientation of pancRNA is important for epigenetic modifications consistent with open chromatin structure. A growing number of noncoding RNAs (ncRNAs) are thought to be involved in sequence-specific alterations of epigenetic processes, mostly causing gene repression. In this study, promoter-associated ncRNAs (pancRNAs >200 nucleotides in size) that were endogenously generated from the sense strand at Map2b, antisense strand at Nefl, and both strands at Vim were investigated regarding their epigenetic potential as positive or negative regulators in rat pheochromocytoma (PC12) and fibroblast (normal rat kidney) cell lines. The respective antisense pancRNAs were associated with several active chromatin marks at the Nefl and Vim promoters. Forced expression of fragments expressing the antisense pancRNAs caused sequence-specific DNA demethylation, whereas a decrease of expression induced methylation of the same sequences. In contrast, perturbing the expression of the two sense pancRNAs did not change the DNA methylation status. These results suggest that a fraction of naturally occurring ncRNAs acts in cis as a single-stranded form and that the transcriptional orientation of pancRNA is important for the establishment of sequence-specific epigenetic modifications consistent with open chromatin structure.

Epigenetic setting and reprogramming for neural cell fate determination and differentiation

In the mammalian brain, epigenetic mechanisms are clearly involved in the regulation of self-renewal of neural stem cells and the derivation of their descendants, i.e. neurons, astrocytes and oligodendrocytes, according to the developmental timing and the microenvironment, the ‘niche’. Interestingly, local epigenetic changes occur, concomitantly with genome-wide level changes, at a set of gene promoter regions for either down- or upregulation of the gene. In addition, intergenic regions also sensitize the availability of epigenetic modifiers, which affects gene expression through a relatively long-range chromatinic interaction with the transcription regulatory machineries including non-coding RNA (ncRNA) such as promoter-associated ncRNA and enhancer ncRNA. We show that such an epigenetic landscape in a neural cell is statically but flexibly formed together with a variable combination of generally and locally acting nuclear molecules including master transcription factors and cell-cycle regulators. We also discuss the possibility that revealing the epigenetic regulation by the local DNA–RNA–protein assemblies would promote methodological innovations, e.g. neural cell reprogramming, engineering and transplantation, to manipulate neuronal and glial cell fates for the purpose of medical use of these cells.

Constrained vertebrate evolution by pleiotropic genes

Despite morphological diversification of chordates over 550 million years of evolution, their shared basic anatomical pattern (or ‘bodyplan’) remains conserved by unknown mechanisms. The developmental hourglass model attributes this to phylum-wide conserved, constrained organogenesis stages that pattern the bodyplan (the phylotype hypothesis); however, there has been no quantitative testing of this idea with a phylum-wide comparison of species. Here, based on data from early-to-late embryonic transcriptomes collected from eight chordates, we suggest that the phylotype hypothesis would be better applied to vertebrates than chordates. Furthermore, we found that vertebrates’ conserved mid-embryonic developmental programmes are intensively recruited to other developmental processes, and the degree of the recruitment positively correlates with their evolutionary conservation and essentiality for normal development. Thus, we propose that the intensively recruited genetic system during vertebrates’ organogenesis period imposed constraints on its diversification through pleiotropic constraints, which ultimately led to the common anatomical pattern observed in vertebrates.Basic anatomical patterns are conserved in chordates. Here, the authors show mid-embryonic conservation during vertebrates’ development and evolutionary constraints introduced by recruitment of mid-embryonic programmes to later stages of development.

Cell - to Species-Level Diversity of Epigenetic Setting for Androgen Receptor Expression in Mammals

During mammalian development, androgen circulates throughout the body and masculinizes several tissues through endocrinological pathways by binding androgen receptor (AR). At the onset of brain masculinization/defeminization, the androgen-AR system functions in a region-specific manner and, even in adulthood, this system affects the transcription of a certain set of genes. The androgen-AR system, together with several coregulators such as histone modifiers, epigenetically regulates many kinds of genes to express the phenotype of a cell according to the cell’s own androgensensitivity as well as the dose of androgen to which it is exposed. Long-range DNA-protein interactions via chromatin looping structures also set up epigenetic regulatory mechanisms that affect the androgen responsiveness. Importantly, the androgen-AR system regulates the transcription of AR itself. For such autoregulation, there are a variety of ciselements within the coding sequences as well as in the regulatory region of AR, including multiple androgen response elements. We found that some of these cis-elements diverged across species: among them there are several primatespecific regions and rodent-specific regions including a short interspersed element, called B2 SINE, as shown by comparisons between primates and rodents. These data suggest that the gain and/or loss of cis-elements by deletion, insertion and mutation determines the species-specific regulation of AR transcription. Differences in the sequences of AR/Ar regulatory regions may contribute to species-specific transcription regulation in genetic and epigenetic manners. Studies focusing on the biodiversity of the AR regulatory region are important for understanding the diversity of the epigenetic setting determining the responsiveness of cells to androgen.

Roles of Epigenetics in the Neural Stem Cell and Neuron

For higher-order functions of the mammalian brain such as the regulation of motor behavior, consciousness, emotion, learning, and memory, neurons have to establish complicated and elaborate networks. In addition, the functions of neurons are critically supported by glial cells (astrocytes and oligodendrocytes). All of these neural cell types (i.e., neurons, astrocytes, and oligodendrocytes) are generated from common neural stem cells (NSCs), which also have self-renewal activity. Accumulating evidence suggests that the behavior of NSCs is influenced spatiotemporally by both cell-extrinsic factors, including cytokine signaling, and cell-intrinsic epigenetic changes, which together regulate the proliferation and fate decisions of NSCs to produce glial cells or neurons, including different neuronal subtypes, in a spatiotemporal manner. In the first half of this chapter, we summarize recent advances in elucidating the role of epigenetic control in the differentiation of NSCs. In postmitotic neurons, as well as NSCs, several orchestrated epigenetic mechanisms underlie neuronal functioning critical for memory formation. Recent studies have revealed the presence and physiological significance of changes of the epigenetic modifications within a neuron of an already defined cell fate. A dynamic change of epigenetic status induced by neuronal activity can alter synaptic plasticity, which constitutes part of the mechanisms of learning and memory. In the latter half of the chapter, we describe the role of epigenetic plasticity in non-dividing neurons. We also discuss the robust identity of the neuronal cell fate, as exemplified by the extremely poor ability of neurons to be reprogrammed to pluripotent stem cells.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos

BackgroundUnderstanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this “mid-embryonic lethality hypothesis” by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations.ResultsBy subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species.ConclusionIn opposition to the “mid-embryonic lethality hypothesis,” our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.