B2 SINE Copies Serve as a Transposable Boundary of DNA Methylation and Histone Modifications in the Mouse.

More than one million copies of short interspersed elements (SINEs), a class of retrotransposons, are present in the mammalian genomes, particularly within gene-rich genomic regions. Evidence has accumulated that ancient SINE sequences have acquired new binding sites for transcription factors (TFs) through multiple mutations following retrotransposition, and as a result have rewired the host regulatory network during the course of evolution. However, it remains unclear whether currently active SINEs contribute to the expansion of TF binding sites. To study the mobility, expression, and function of SINE copies, we first identified about 2,000 insertional polymorphisms of SINE B1 and B2 families within Mus musculus. Using a novel RNA sequencing method designated as melRNA-seq, we detected the expression of SINEs in male germ cells at both the subfamily and genomic copy levels: the vast majority of B1 RNAs originated from evolutionarily young subfamilies, whereas B2 RNAs originated from both young and old subfamilies. DNA methylation and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses in liver revealed that polymorphic B2 insertions served as a boundary element inhibiting the expansion of DNA hypomethylated and histone hyperacetylated regions, and decreased the expression of neighboring genes. Moreover, genomic B2 copies were enriched at the boundary of various histone modifications, and chromatin insulator protein, CCCTC-binding factor, a well-known chromatin boundary protein, bound to >100 polymorphic and >10,000 non-polymorphic B2 insertions. These results suggest that the currently active B2 copies are mobile boundary elements that can modulate chromatin modifications and gene expression, and are likely involved in epigenomic and phenotypic diversification of the mouse species.

melRNA-seq for Expression Analysis of SINE RNAs and Other Medium-Length Non-Coding RNAs.

BACKGROUND: Small interspersed elements (SINEs) are transcribed by RNA polymerase III (Pol III) to produce RNAs typically 100-500 nucleotides in length. Although their RNA abundance can be evaluated by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs or SINE sequences present in longer transcripts. RESULTS: To elucidate the abundance, source loci, and sequence nature of SINE RNAs, we established a deep sequencing method, designated as melRNA-seq (medium-length RNA-seq), which can determine whole-length RNA sequences. Total RNA samples were treated with 5' pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5' end of intact SINE RNAs. Similarly, another adaptor was ligated to the 3' end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. The analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of SINE expression data both at family and locus levels. CONCLUSIONS: This new method can be used for quantification and detailed sequence analysis of medium-length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. Further, its dynamic range is much wider than Northern blotting and primer extension.

The effects of a switch-off response accompanied by hypothalamically induced restlessness on immunoendocrinological changes in cats.

Electrical stimulation of the anterior hypothalamus in cats elicits a behavior called restlessness. When a switch is available for the cats to shut off the electrical stimulation, the cats learn to turn off the stimulation (switch-off response; SOR). In this study, we examined the relationship between the SOR and immunoendocrinological alterations. First of all, an escapable stimulation, in which cats could turn off the stimulation, was applied (escapable condition; EC). One month later, inescapable stimulation was delivered under the same conditions except for the fact that the cats could not turn off the stimulation (inescapable condition; IC). A behavioral analysis revealed that unstable patterns of behavior and a reduction in motor activity were observed in IC compared with those in EC. Furthermore, no significant changes in peripheral leukocytes were observed, while plasma epinephrine and cortisol transiently increased after the series of stimulations, but immediately decreased after the end of the stimulation in EC. On the other hand, there was a greater and prolonged increase in the number of peripheral granulocytes and the plasma levels of epinephrine and cortisol from 1 to 2 h after the stimulation until the end of the experiment in IC. Regarding the number of peripheral lymphocytes, CD4+ or CD8+ lymphocytes and the CD4+ to CD8+ ratio, no significant differences were found between EC and IC. These results suggest that the inability to escape from the aversive stimulation caused a decrease in movement and a prolonged alteration of the immune and endocrine systems, as is often observed in learned helplessness.