Determination of the n3β-d structure.

<p>(a) The time-domain DEER data for the n3β-d (0.5 mM) in the studied conditions, including the vitrified bulk solvent (sol(s)/H₂O) and the nanochannels (SBA15a and SBA15b). The gray lines represent the exponential baselines that best fit the DEER data. There are two insets. One displays a ribbon model for the n3β-d showing the spin-label side-chains at the 3rd and 9th sites of the peptide. The model was derived from a NMR study (PDB code: 1G04). The other inset shows the baseline-corrected DEER traces for the sol(s)/H₂O and the SBA15a, and also the simulated DEER traces (in green color) using the obtained P(r)s. There are some distinct differences in the two traces. (b) The (normalized) interspin distance distributions of the n3β-d peptides in the conditions studied. The average distances of the three measurements are approximately the same, indicating the n3β structure remains roughly unchanged. A much-broadened P(r) for the bulk solution study is obtained due to the solvent heterogeneity. The inset shows the Pake doublets converted from the DEER data. (c) Cw-ESR spectra of the n3β-d at 50 K. The clustering, caused by the solvent heterogeneity at 50 K, is evidently observed in the cw-ESR spectra of the bulk solution study, but not in the nanochannel studies.</p

Water accessibility study of the PPm3-s by ESEEM.

<p>(a) Three-pulse ESEEM time-domain data (solid lines) after the removal of the exponential decaying function in the raw data. The modulation depth is directly correlated to the peak intensity of the FT-ESEEM and can be quantitatively characterized by the best-fit parameter <i>k_D</i> (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>). The dashed lines represent the theoretical fits to the experimental data using the equation described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. (b) The FT-ESEEM data for the PPm3-s in various deuterated conditions. The peaks correspond to the Larmor frequency of nucleus ²H, indicating the PPm3-s is surrounded by D₂O. The inset shows a ribbon model of a PPm3 variant carrying three spin labels.</p

Parameters obtained in the analyses of the ESE and ESEEM data.^§

§<p>Estimated errors: 5%(T_M), 10%(x), 13% (<i>C_ex</i>), 5% (<i>k_D</i>), 10% (Π). Abbreviations: <b>n3-s-a</b> (the n3-s is within SBA15a containing pure water); <b>n3-s-b</b> (the n3-s is within SBA15b containing pure water); <b>n3-s-sol(s)</b> (the n3-s is in a vitrified bulk solvent containing 40 wt% sucrose, (s), in D₂O or H₂O); <b>PPm3-s-sol(g)</b> (PPm3-s is in a vitrified bulk solvent containing 40v/v% glycerol in H₂O; deuterated glycerol is used if the solvent is D₂O, a condition of which is represented by sol(dg)/D₂O in main text); <b>PPm3-s-sol(s)</b> (PPm3-s is in a vitrified bulk solvent containing 40 wt% sucrose in D₂O or H₂O). In all of the experiments, the surface group of the nanochannels is modified to –SiOD in advance if D₂O is used. See Method for details.</p>#<p>The values of T_M and x are obtained in the analysis of the pulsed ESE measurements using a stretched exponential function, , where τ is the time between the two pulses, x the exponent, and Y(0) is the echo intensity at τ  = 0. The obtained values are used to yield <i>C_ex</i> using Eq. (2). The <i>C_ex</i> represents ESE-based water accessibility within the range of ∼2 nm from the nitroxide spin.</p>¶<p>The <i>k_D</i> values are obtained in the theoretical analysis of the ESEEM measurements as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. The best-fit values for the damping constant (<i>τ₀</i>) and phase (φ) are very close together (2.9∼3.0). The Π represents ESEEM-based water accessibility within the range of ∼0.35 nm from the nitroxide spin.</p

Determination of the n3α-d structure.

<p>(a) The time-domain DEER signals of the studied conditions. The gray lines represent the exponential baselines that best fit the data. Inset shows a ribbon model of the n3α-d derived from NMR data (PDB code: 1M25). (b) The P(r) distributions extracted from the time-domain DEER data by the Tikhonov regularization analysis. The average distances (∼2.0 nm) are consistent with the expectation. (c) The cw-ESR spectra of the n3α-d. The spectra of the bulk solution studies are characterized by a broader linewidth and the spectral heterogeneity (indicated by arrows) as compared to the spectra of the nanochannel studies.</p

5hmC enrichment profile similarity among the 12 mouse samples.

<p>(A) An unsorted similarity matrix of intragenic 5hmC coverage. The colors represent the correlation scores (i.e., similarity) ranging from dark blue (ρ = 0.5) to dark red (ρ = 1.0). (B) Visualization of the genomic region around <i>Xist</i>. The 5hmC-enriched regions for each sample are shown in separate labeled track. The sample names are abbreviated. F and M denote female and male samples respectively. Cb = cerebellum; Cx = cortex; Hi = hippocampus; Hy = hypothalamus; Th = thalamus; Lv = liver.</p

5hmC enrichment in relation to intragenic regions across 12 samples.

<p>We evaluate the intragenic 5hmC enrichment patterns in (A) protein-coding genes, (B) process transcripts, (C) lincRNAs, (D) enzymes, (E) G-protein coupled receptors, (F) kinases (G), others, (H) transcription regulators and (I) transporters. The 5hmC coverage within gene bodies and up to 2.5 kb upstream of transcription start site (TSS) and downstream of transcription termination site (TTS) are shown. Gray box represents the gene body from TSS to TTS. The sample names are abbreviated. Cb = cerebellum; Cx = cortex; Hi = hippocampus; Hy = hypothalamus; Th = thalamus; Lv = liver.</p

Long-range water accessibility study by ESE.

<p>The theoretical fits (red lines) to the ESE experimental data (blue lines) using a stretched exponential function (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>) as previously described. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Huang1" target="_blank">[11]</a> The results for the n3β-s and PPm3-s are shown in (a) and (b), respectively. The decay signals acquired by the ESE experiments were fitted over the maxima of the deuterium modulation as described in Zecevic et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Zecevic1" target="_blank">[32]</a> to minimize the influence from destructive interference of nuclear modulations. The obtained values of the T_M (in ns) and stretching exponent x are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>. The T_M values can be directly used to yield the surrounding proton density (<i>C_ex</i>; cf. Eq. 2) within the range of ∼2 nm from a nitroxide spin.</p

Box plots representation of intragenic 5hmC enrichment versus RNA expression levels of 319 ion channel genes.

<p>The distribution expression intensities of genes with no (0%), low (<20%), intermediary (20 ~ 50%), high (50% ~ 80%), and very high (> 80%) intragenic 5hmC enrichment. The number of genes in each 5hmC enrichment class is labeled above the corresponding box plot. The brain and liver sample are color in red and blue colors respectively. The sample names are abbreviated. F and M denote female and male samples respectively. Cb = cerebellum; Cx = cortex; Hi = hippocampus; Hy = hypothalamus; Th = thalamus; Lv = liver.</p

Correlated 5-Hydroxymethylcytosine (5hmC) and Gene Expression Profiles Underpin Gene and Organ-Specific Epigenetic Regulation in Adult Mouse Brain and Liver

<div><p>Background</p><p>DNA methylation is an epigenetic mechanism essential for gene regulation and vital for mammalian development. 5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. Aside from being a key intermediate in cytosine demethylation, 5hmC may have potential regulatory functions with emerging importance in mammalian biology.</p><p>Methods</p><p>Here, we investigate the global 5hmC enrichment in five brain structures, including cerebellum, cerebral cortex, hippocampus, hypothalamus and thalamus, as well as liver tissues from female and male adult mice by using chemical capture-based technique coupled with next-generation sequencing. At the same time, we carried out total RNA sequencing (RNA-seq) to analyze the transcriptomes of brain regions and liver tissues.</p><p>Results</p><p>Our results reveal preferential 5hmC enrichment in the gene bodies of expressed genes, and 5hmC levels of many protein-coding genes are positively correlated with RNA expression intensity. However, more than 75% of genes with low or no 5hmC enrichment are genes encode for mitochondrial proteins and ribosomal proteins despite being actively transcribed, implying different transcriptional regulation mechanisms of these housekeeping genes. Brain regions developed from the same embryonic structures have more similar 5hmC profiles. Also, the genic 5hmC enrichment pattern is highly tissue-specific, and 5hmC marks genes involving in tissue-specific biological processes. Sex chromosomes are mostly depleted of 5hmC, and the X inactive specific transcript (<i>Xist</i>) gene located on the X chromosome is the only gene to show sex-specific 5hmC enrichment.</p><p>Conclusions</p><p>This is the first report of the whole-genome 5hmC methylome of five major brain structures and liver tissues in mice of both sexes. This study offers a comprehensive resource for future work of mammalian cytosine methylation dynamics. Our findings offer additional evidence that suggests 5hmC is an active epigenetic mark stably maintained after the global reprogramming event during early embryonic development.</p></div

Average intragenic 5hmC coverage of genes classified by (A) gene biotype and (B) molecule type in 12 mouse brain and liver samples.

<p>The categorical data points were joined and shown as line graphs to allow visualization of the 5hmC enrichment trends across biotypes and molecule types among the 12 samples. The sample names are abbreviated. Cb = cerebellum; Cx = cortex; Hi = hippocampus; Hy = hypothalamus; Th = thalamus; Lv = liver.</p