Linker histone variant H1T targets rDNA repeats

<p>H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.</p

MOESM1 of H2A O-GlcNAcylation at serine 40 functions genomic protection in association with acetylated H2AZ or ÎłH2AX

Additional file 1: Figure S1-S3 and Table S1-S2

RNA-Seq Analysis of Equine Conceptus Transcripts during Embryo Fixation and Capsule Disappearance

<div><p>Extensive studies have been conducted to characterize the unique phenomena of equine pregnancy. Most studies have focused on embryo transmigration when the embryo is covered with a mucin-like glycoprotein capsule and on the characterization of the chorionic girdle and chorionic gonadotropin (CG) secretion. However, the events preceding and following capsule disappearance have not been well studied. In this study, the mRNA expression in conceptus membranes at days 19, 21, and 25 (day 0 = day of ovulation) was analyzed by RNA-seq (SOLiD3), and transcript levels on these three days and day 13 were confirmed by real-time PCR. Of the 26,416 equine genes registered, 20,436 transcripts were aligned to sequences in the Ensembl database, from which 4,625 transcripts were registered in both Ensembl and the KEGG pathway. Each of the 4,625 transcripts was examined through KEGG pathway analysis, and 12 transcripts of integrins (<i>ITG</i>s) and collagens (<i>COL</i>s) were confirmed through real-time PCR. Our data indicated that extracellular matrix (ECM)-related mRNAs were highly expressed in day 19, 21, and 25 conceptus membranes. In combination with previous results, which confirmed a lack of laminin and fibronectin transcript expression in the endometrium, these observations suggest that in contrast to attachment through focal adhesion, conceptus chorionic membrane ECMs function as a scaffold-like structure to possibly maintain the shape of the conceptus and a separation between chorionic membranes and the uterine luminal epithelium.</p></div

Reactivation of hyperglycemia-induced hypocretin (<i>HCRT)</i> gene silencing by <i>N</i>-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells

<p>Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with <i>N</i>-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-<i>O</i>-GlcNAcylation on the <i>HCRT</i> gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the <i>HCRT</i> gene, which was characterized by H3/H4 hypoacetylation and hyper-<i>O</i>-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the <i>HCRT</i>-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the <i>HCRT</i> silencing. Thus, the epigenetic status of the <i>HCRT</i> gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated <i>HCRT</i> gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the <i>HCRT</i> gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.</p

Collagen transcript analysis by qPCR vs. RNA-seq.

<p>Total RNA was isolated from whole day 13 conceptuses, while for day 19, 21, and 25 conceptuses, yolk sac fluid was carefully removed and heart and blood vessel structures were separated from extraembryonic membranes. The extraembryonic membrane samples consisted of yolk sac and chorion on day 19 and 21 whereas on day 25, the samples contained yolk sac and allantochorion membranes. Collagen transcripts analyzed were <i>COL4A1</i>, <i>COL4A5</i>, <i>COL4A6</i>, <i>COL5A2</i>, <i>COL6A1</i>, <i>COL6A2</i>, and <i>COL6A3</i>. Gray bars indicate data from RNA-seq analysis, and dark bars represent data from qPCR analyses. Note that RNA-seq data represent transcripts extracted from day 19, 21 and 25 trophectoderm and/or chorionic membrane, whereas RNA from days 13, 19, 21 and 25 were analyzed by qPCR. An average of at least three conceptuses was analyzed for each time point, and the SEM is shown. Please note that values on Y-axis resulted from the generation of a standard curve for each transcript. In doing so, the highest amount in each transcript was treated as “1”, and relative values were presented.</p

Oligonucleotide primers for RT-PCR and real-time PCR analyses.

<p>F: Forward, R: Reverse.</p><p>Oligonucleotide primers for RT-PCR and real-time PCR analyses.</p

ECM receptor pathway analysis; integrin transcripts.

<p>Pathway analysis was extended to the expression of integrin transcripts. There are three columns, each containing potential ligands and integrin receptor heterodimers. αVβ3 is on the upper right: αV transcripts were high on day 21 and decreased toward day 25.</p

Focal adhesion pathway analysis.

<p>Identified transcripts were aligned to the Ensembl equine database and analyzed through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (<a href="http://www.genome.ad.jp/kegg/pathway.html" target="_blank">http://www.genome.ad.jp/kegg/pathway.html</a>). Because we expected that scaffold-like structures form during these time points, the first KEGG analysis examined focal adhesion; cytokine-cytokine interactions and ECM-receptor interactions. Data represent transcripts of day 21 and 25 conceptuses. There are more than two layers, i.e., Caveolin transcripts were found in the cell membrane on top <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114414#pone.0114414-Mohanty1" target="_blank">[43]</a>. Yellow color on the top-left side of box indicates the transcript of one of the Caveolin variants increased on day 21 (vs. day 19). White color on the left and right of the bottom indicates that the transcripts related to the second Caveolin variant were not found on days 21 or 25 in this RNA-seq analysis.</p

ECM receptor pathway analysis; collagen, laminin, fibronectin, vitronectin, osteopontin, von Willebrand factor, chondroadherin, and reelin transcripts.

<p>Pathway analysis was further extended to the expression of collagen, laminin, fibronectin, vitronectin, osteopontin, von Willebrand factor, chondroadherin, and reelin transcripts. <i>COL4A3</i> transcripts are on the left in yellow and on the right in pink, indicating that <i>COL4A3</i> expression is nearly 5-fold higher on day 21 than day 19, and further increased on day 25.</p

Changes in integrin transcripts, qPCR vs. RNA-seq.

<p>Total RNA was isolated from whole day 13 conceptuses, while for day 19, 21, and 25 conceptuses, yolk sac fluid was carefully removed and heart and blood vessel structures were separated from extraembryonic membranes. The extraembryonic membrane samples consisted of yolk sac and chorion on day 19 and 21 whereas on day 25, the samples contained yolk sac and allantochorion membranes. Integrin transcripts analyzed were <i>ITGA4</i>, <i>ITGA5</i>, <i>ITGA6</i>, <i>ITGB4</i> and <i>ITGB5</i>. Gray bars indicate data from RNA-seq analysis, and dark bars represent data from qPCR analyses. Note that RNA-seq data represent transcripts extracted from day 19, 21 and 25 trophectoderm and/or chorionic membrane, whereas RNA from days 13, 19, 21 and 25 were analyzed by qPCR. An average of at least three conceptuses was analyzed for each time point, and the SEM is shown. Please note that values on Y-axis resulted from the generation of a standard curve for each transcript. In doing so, the highest amount in each transcript was treated as “1”, and relative values were presented.</p