Tissue-specific gene expression concomitant with intragenic 5hmC enrichment.

<p>Box plots showing the distribution of proportion of gene bodies that overlap 5hmC-enriched peaks in genes expressed only in (A) brain and (B) liver samples. Each point represents a tissue-specific gene, and there are 331 brain-specific genes shown in (A) and 124 liver-specific genes shown in (B). 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. There were significant differences in 5hmC enrichment levels between the two tissue groups assessed by one-way ANOVA in both tissue-specific gene sets (<i>P</i> < 0.05).</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

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

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

The expression intensities of Tet genes and 5hmC readers.

<p>Bar plots showing the average RNA expression levels of <i>Tet1</i>, <i>Tet2</i>, <i>Tet3</i> and 22 5hmC readers represented as read count-per-million. Error bars show the 95% confidence intervals of the means. Statistical differences between brain and liver samples are indicated with asterisks (* <i>P</i> < 1e-2; ** <i>P</i> < 1e-3; *** <i>P</i> < 1e-5; Two Sample t-test). 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 statistics in mouse brain and liver tissues.

<p>(A) The distribution of the size of 5hmC enriched regions in the 12 samples. (B) The percentage genome that is 5hmC-enriched. The per-chromosome 5hmC enrichment of the (C) intragenic regions and (D) intergenic regions in brain and liver samples. 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

Box plots representation of RNA expression levels of genes classified by (A) gene biotypes and (B) molecule types in the 12 mouse brain and liver samples.

<p>The box plots show medians and interquartile ranges of the expression intensities. The red and blue colors represent genes with and without intragenic 5hmC enrichment respectively. Statistical differences between genes with and without 5hmC in the nine biotypes are indicated with asterisks (* <i>P</i> < 0.05; ** <i>P</i> < 1e-5; *** <i>P</i> < 1e-10; Wilcoxon rank sum test). All molecule types showed significance statistical differences (<i>P</i> < 1e-5; Wilcoxon rank sum test).</p

Intragenic 5hmC enrichment patterns and expression intensities of five clusters of protein-coding genes.

<p>(A) The protein-coding genes are classified into five clusters according to 5hmC enrichment patterns within gene body (from TSS to TTS). The clusters are arranged with increase 5hmC coverage from C1 to C5. The number of genes in each cluster is given in each subplot. (B) The box plots representation of RNA expression levels of genes in each cluster in the 12 mouse brain and liver samples. 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

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