Transposons contribute to the acquisition of cell type-specific cis-elements in the brain.

Mammalian brains have evolved in stages over a long history to acquire higher functions. Recently, several transposable element (TE) families have been shown to evolve into cis-regulatory elements of brain-specific genes. However, it is not fully understood how TEs are important for gene regulatory networks. Here, we performed a single-cell level analysis using public data of scATAC-seq to discover TE-derived cis-elements that are important for specific cell types. Our results suggest that DNA elements derived from TEs, MER130 and MamRep434, can function as transcription factor-binding sites based on their internal motifs for Neurod2 and Lhx2, respectively, especially in glutamatergic neuronal progenitors. Furthermore, MER130- and MamRep434-derived cis-elements were amplified in the ancestors of Amniota and Eutheria, respectively. These results suggest that the acquisition of cis-elements with TEs occurred in different stages during evolution and may contribute to the acquisition of different functions or morphologies in the brain.

Binding patterns of RNA-binding proteins to repeat-derived RNA sequences reveal putative functional RNA elements.

Recent reports have revealed that repeat-derived sequences embedded in introns or long noncoding RNAs (lncRNAs) are targets of RNA-binding proteins (RBPs) and contribute to biological processes such as RNA splicing or transcriptional regulation. These findings suggest that repeat-derived RNAs are important as scaffolds of RBPs and functional elements. However, the overall functional sequences of the repeat-derived RNAs are not fully understood. Here, we show the putative functional repeat-derived RNAs by analyzing the binding patterns of RBPs based on ENCODE eCLIP data. We mapped all eCLIP reads to repeat sequences and observed that 10.75 % and 7.04 % of reads on average were enriched (at least 2-fold over control) in the repeats in K562 and HepG2 cells, respectively. Using these data, we predicted functional RNA elements on the sense and antisense strands of long interspersed element 1 (LINE1) sequences. Furthermore, we found several new sets of RBPs on fragments derived from other transposable element (TE) families. Some of these fragments show specific and stable secondary structures and are found to be inserted into the introns of genes or lncRNAs. These results suggest that the repeat-derived RNA sequences are strong candidates for the functional RNA elements of endogenous noncoding RNAs.

Association analysis of repetitive elements and R-loop formation across species.

BACKGROUND: Although recent studies have revealed the genome-wide distribution of R-loops, our understanding of R-loop formation is still limited. Genomes are known to have a large number of repetitive elements. Emerging evidence suggests that these sequences may play an important regulatory role. However, few studies have investigated the effect of repetitive elements on R-loop formation. RESULTS: We found different repetitive elements related to R-loop formation in various species. By controlling length and genomic distributions, we observed that satellite, long interspersed nuclear elements (LINEs), and DNA transposons were each specifically enriched for R-loops in humans, fruit flies, and Arabidopsis thaliana, respectively. R-loops also tended to arise in regions of low-complexity or simple repeats across species. We also found that the repetitive elements associated with R-loop formation differ according to developmental stage. For instance, LINEs and long terminal repeat retrotransposons (LTRs) are more likely to contain R-loops in embryos (fruit fly) and then turn out to be low-complexity and simple repeats in post-developmental S2 cells. CONCLUSIONS: Our results indicate that repetitive elements may have species-specific or development-specific regulatory effects on R-loop formation. This work advances our understanding of repetitive elements and R-loop biology.

The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

A noncoding RNA regulates the neurogenin1 gene locus during mouse neocortical development.

The proneural basic helix-loop-helix (bHLH) transcription factor neurogenin1 (Neurog1) plays a pivotal role in neuronal differentiation during mammalian development. The spatiotemporal control of the Neurog1 gene expression is mediated by several specific enhancer elements, although how these elements regulate the Neurog1 locus has remained largely unclear. Recently it has been shown that a large number of enhancer elements are transcribed, but the regulation and function of the resulting transcripts have been investigated for only several such elements. We now show that an enhancer element located 5.8-7.0 kb upstream of the mouse Neurog1 locus is transcribed. The production of this transcript, designated utNgn1, is highly correlated with that of Neurog1 mRNA during neuronal differentiation. Moreover, knockdown of utNgn1 by a corresponding short interfering RNA inhibits the production of Neurog1 mRNA in response to induction of neuronal differentiation. We also found that production of utNgn1 is suppressed by polycomb group (PcG) proteins, which inhibit the expression of Neurog1. Our results thus suggest that a noncoding RNA transcribed from an enhancer element positively regulates transcription at the Neurog1 locus.

Formation of a FGF-2 and calcium phosphate composite layer on a hydroxyapatite ceramic for promoting bone formation.

Fibroblast growth factor-2 (FGF-2) was immobilized on a hydroxyapatite (HAP) ceramic in supersaturated calcium phosphate solution prepared using solutions corresponding to clinically approved infusion fluids. To avoid the risk of FGF-2 denaturation, FGF-2 immobilization was carried out at 25 degrees C. FGF-2 was successfully immobilized on HAP ceramic surfaces by deposition with calcium phosphate to form a FGF-2 and calcium phosphate composite layer. A maximum of 2.72 +/- 0.01 microg cm(-2) of FGF-2 was immobilized in the composite layer formed on the HAP ceramic under the optimum condition. A FGF-2-immobilized HAP ceramic is likely to have the ability to release a sufficient amount of FGF-2 to promote bone formation. FGF-2 released from a FGF-2-immobilized HAP ceramic maintained its biological activity, since the proliferation of fibroblastic NIH3T3 was promoted. Therefore, the FGF-2-immobilized HAP ceramic is expected to be a useful material for promoting new bone formation.

Formation of an ascorbate-apatite composite layer on titanium.

An ascorbate-apatite composite layer was successfully formed on NaOH- and heat-treated titanium by coprecipitating L-ascorbic acid phosphate and low-crystalline apatite in a supersaturated calcium phosphate solution at 37 degrees C for 48 h. The supersaturated calcium phosphate solutions used have chemical compositions attainable by mixing infusion fluids officially approved for clinical use. The amount of immobilized L-ascorbic acid phosphate ranged from 1.0 to 2.3 microg mm(-2), which is most likely to be sufficient for the in vitro osteogenic differentiation of mesenchymal stem cells on titanium. Since ascorbate is important for the collagen synthesis and subsequent osteogenesis of mesenchymal stem cells, titanium coated with the ascorbate-apatite composite layer would be useful as a scaffold in bone tissue engineering and as a bone substitute.