Genome-wide analysis of H3.3 nucleosome turnover.

<p>(A) Experimental scheme to determine the turnover index. (B) Two-dimensional histogram of <i>T₂₄</i> and <i>T₄₈</i> for all H3.3 nucleosomes. (C) Distribution profiles of the H3.3 nucleosome turnover index around the TSS (left panel) and TES (right panel). (D) Bimodal distribution of turnover at +1 nucleosome versus expression level. Genes were sorted by RPKM from high to low with a sliding widow of 600 genes and then plotted against their turnover index at the +1 nucleosome. (E) Genomic distribution of high turnover and low turnover H3.3 nucleosomes.</p

H3.3 nucleosomes with higher turnover index tend to associate with higher splitting index.

<p>(A) The distribution of splitting index for H3.3 nucleosomes within each specified turnover index range. (B) The distribution of splitting index for H3.3 nucleosomes within the highest turnover index ranges.</p

The enhancer H3.3 nucleosomes display higher splitting index than the non-enhancer H3.3 nucleosomes.

<p>(A) Box plot of the splitting index of enhancer or non-enhancer H3.3 nucleosomes within the same turnover ranges. (B) Box plot of the splitting index of H3.3 nucleosomes at the enhancers, promoters, 5-UTRs within the same turnover ranges. (C) Percentage of split nucleosomes for enhancer H3.3 or non-enhancer H3.3 at various turnover ranges. *** indicates the significant difference with P value<0.0001. (D) Dual-tagged H3.3 nucleosomes derived from the co-expression experiment did not show enrichment at cell-type specific enhancers.</p

H3.3 nucleosome splitting events are better markers for active transcription than H3.3 nucleosome occupancy.

<p>(A) Split H3.3 nucleosomes were enriched in the top 25% expression level genes, as compared to the total H3.3 nucleosomes or non-split H3.3 nucleosomes. Non-split H3.3 nucleosomes were enriched in the bottom 25% expression level genes. P values were calculated with chi-square test. ***P<0.001, **P<0.01, #P>0.1. (B) After normalization against the H3.3 occupancy, the split but not the non-split H3.3 nucleosomes were enriched at active genes. H3.3 nucleosomes at the entire genes were analyzed together.</p

Moderate correlation between the H3.3 turnover index and splitting index.

<p>(A) Turnover index distribution profile for all H3.3 nucleosomes. (B) Turnover index distribution profile for the split H3.3 nucleosomes. (C) Turnover index distribution profile for the non-split H3.3 nucleosomes. (D) Box plot for the turnover index of all, split, and non-split H3.3 nucleosomes.</p

H3.3 nucleosome splitting events feature cell-type specific enhancers.

<p>(A) An example enhancer region enriched with split H3.3 nucleosomes. Profiles of single-round ChIPs, sequential ChIP, turnover index, splitting index are illustrated. Percentile ranking of turnover index and splitting index are shown in a grey scale. (B) Split H3.3 nucleosomes were specifically enriched at enhancers, whereas the non-split H3.3 nucleosomes were specifically depleted at enhancers. (C) Distribution of the H3.3 nucleosomes, split and non-split H3.3 nucleosomes, intergenic split H3.3 nucleosomes and high and low turnover H3.3 nucleosomes at the cell-type specific enhancers. (D) All H3.3 nucleosomes were sorted by their splitting index and grouped into 5000 nucleosome widows. These nucleosomes were then plotted against their overlap percentage with enhancers. The arbitrarily defined split and non-split nucleosomes with top or bottom 5% splitting index were boxed in red. (E) Similar to (D), but common enhancers were excluded. (F) The 10-kb genomic intervals sorted by their numbers of split nucleosomes were plotted against their overlap percentage with the cell-type specific enhancers. (G) Similar to (F), but common enhancers were excluded.</p

Determine H3.3 nucleosome occupancy, turnover and splitting events at the genome-wide level.

<p>(A) Induction of Flag-H3.3 and HA-H3.3 histones. (B) Experimental scheme to determine the splitting index. (C) Distribution profiles of new H3.3 nucleosomes (Flag-tagged) around the TSS (left panel) and TES (right panel). Genes were divided into 3 groups according to their RPKM: High, the top one-third genes; Medium, the middle one-third genes and Low, the bottom one-third genes. (D) Distribution profiles of old H3.3 nucleosomes (HA-tagged).</p

Expression dynamics, relationships, and transcriptional regulations of diverse transcripts in mouse spermatogenic cells

<p>Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.</p