Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression

<div><p>Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes <i>in vivo</i>. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene <i>Nr1d1</i> in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure.</p></div

Global analysis of cohesin and CTCF on circadian transcription.

<p>(a) The distribution of circadian rhythmicity of transcription around the protein binding meta-sites (Methods). The circadian index was defined from the negative strand of GRO-Seq [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref009" target="_blank">9</a>]. The result from positive strand was shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.s003" target="_blank">S3A Fig</a>. The data points are connected by spline smoothing method. The circadian rhythmicity is high around Bmal1, Nr1d1, and cohesin-non-CTCF sites but not around cohesin-CTCF sites. (b) Both Bmal1 and Nr1d1 binding sites have significantly higher overlaps with cohesin-non-CTCF sites (CNC) than cohesin-CTCF (CC) sites in mouse liver (Fisher’s exact test p = 10⁻¹⁶). (c) The distributions of phase differences of neighboring COGs are shown in a violin plot. The phase differences across Bmal1 and Nr1d1 binding sites are significantly smaller than the genomic background, whereas the phase differences across cohesin-CTCF sites are larger than the genomic background (Mann-Whitney U test). (d) The violin plot of phase variance in cohesin-CTCF domains. The medium and large cohesin-CTCF domains inferred from cohesin ChIA-PET data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref029" target="_blank">29</a>] have smaller phase variances than the background domains (Mann-Whitney U test). ****p < 10⁻⁸,**p < 0.01, *p < 0.05.</p

The candidate COGs interacting with circadian enhancers via invariant cohesin-mediated loops.

<p>The candidate COGs interacting with circadian enhancers via invariant cohesin-mediated loops.</p

The expression of a COG, <i>Phldb2</i>, is influenced by an invariant cohesion-mediated enhancer-promoter interaction.

<p>(a) A cohesin loop connects the BMAL1 enhancer (marked by asterisk) and the promoter of <i>Phldb2</i>. Conserved cohesin binding sites and active histone marks between liver and MEFs were found at the promoter of <i>Phldb2</i> and its enhancer. The enhancer was bound by Bmal1 in liver but not in MEFs. The locations of primers used in 3C assay in liver and MEFs were indicated below the RefGenes. ∆C denotes CRISPR-mediated deletion region. The normalized RNA-Seq in <i>Smc3</i>-/- and control MEFs in this region were showed in the bottom. (b) The 3C signals of interactions anchored to <i>Phldb2</i> promoter in mouse liver (ANOVA p = 10⁻⁷, mean+/-SD, 2 biological replicates, 4 technical replicates). CN, control. EN, enhancer. (c) The 3C signals of <i>Phldb2</i> promoter-enhancer interaction in control and <i>Smc3</i>-/- MEFs (t-test p = 0.04, mean+/-SD, 2 biological replicates, 3 technical replicates). (d) The normalized expression level of <i>Phldb2</i> after the deletion of cohesin binding site near the enhancer of <i>Phldb2</i> in Hepa1-6 cells (t-test p = 10⁻¹¹, mean+/-SD, 2 biological replicates, 3 technical replicates). ***p < 10⁻⁴, *p < 0.05.</p

A model of circadian regulation of gene expression under high-order chromosome organization.

<p>(a) Cohesin-CTCF disrupts the gene regulation of circadian transcription factors, whereas cohesin-non-CTCF facilitates the contacts between circadian enhancers and promoters. (b) In the background model, only Bmal1 binding sites within 2 Mb of the genes were considered. In the cohesin/CTCF dependent model, the contribution of each Bmal1 binding site was further multiplied by three cohesion/CTCF dependent factors: <i>CNC</i>_<i>i</i>, <i>CNC</i>_<i>j</i>, and <i>CC</i>_<i>ij</i> (Methods). (c) The density plot of regulatory potentials in COGs or non-COGs under different conditions. The COGs in cohesin/CTCF dependent model, as well as in the model with only cohesin-CTCF or cohesin-non-CTCF effect, have significantly higher Bmal1 regulatory potentials than the background model (Kolmogorov-Smirnov test p = 10⁻¹⁶). The regulatory potential of the model where cohesin-non-CTCF sites and cohesin-CTCF sites were interchanged is similar with the background model. (d) The density plot of phases of COGs top ranked in two models. The enriched phase regime (CT6-CT12, dash lines) of COGs with top 10% Bmal1 regulatory potentials in cohesin/CTCF dependent model follows the peak of Bmal1 binding at CT6. (e) The density plots of differentially expressed genes in <i>Bmal1</i> and <i>Nr1d1</i> KO datasets. Under-expressed genes in <i>Bmal1</i> KO (Fan et al., manuscript in preparation) have higher Bmal1 regulatory potentials in cohesin/CTCF dependent model than those in the background model, whereas over-expressed genes in <i>Nr1d1</i> KO [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref009" target="_blank">9</a>] have markedly increased in Nr1d1 regulatory potentials.</p

Cohesin is involved in circadian interactome.

<p>(a) The COGs in mouse liver interacting with the enhancer at CT6 or CT18. Their circadian phases and distances to the bait were listed. (b) The enhancer interacting regions significantly overlap more with the binding sites of cohesin-non-CTCF than cohesin-CTCF and randomly permutated cohesin-CTCF or cohesin-non-CTCF sites at both time points (binomial test). These two binding sites were identified from <i>de novo</i> analysis of ChIP-Seq data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref022" target="_blank">22</a>] (Methods). (c) The H3K4me1, H3K27ac [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref031" target="_blank">31</a>], cohesin, and CTCF [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref022" target="_blank">22</a>] ChIP-Seq signals around 97 Bmal1 super-enhancers. The binding sites were sorted by the signals on Bmal1 ChIP-Seq data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005992#pgen.1005992.ref006" target="_blank">6</a>]. The Bmal1 super-enhancers contain higher binding signals of cohesin than CTCF. ***p < 10⁻⁴.</p

Cohesin knockout in MEFs.

<p>(a) The RT-PCR results of the expression of clock genes in control (CN) and <i>Smc3-/-</i> (KO) MEFs. Gene expression were measured every 4 hr for 24 hr after the cells were synchronized by 1-hr dexamethasone treatment at CT0. Each data point contained two biological replicates and three technical replicates. All four clock genes showed significant circadian expression in CN and KO by cosine fitting (p < 0.05) except for <i>Nr1d1</i> in KO. ANOVA analysis showed that the expression of <i>Nr1d1</i> has significant difference between CN and KO (p = 10⁻⁷). (b) The scatterplot of global gene expression from RNA-Seq in <i>Smc3</i>-/- and control MEFs. 20-hr and 32-hr samples were combined together to determine the differentially expressed genes. Core clock genes including <i>Clock</i>, <i>Rora</i>, and <i>Npas2</i> were under-expressed in cohesin KO.</p

Additional file 1 of Apoptotic extracellular vesicles derived from hypoxia-preconditioned mesenchymal stem cells within a modified gelatine hydrogel promote osteochondral regeneration by enhancing stem cell activity and regulating immunity

Additional file 1. Fig.S1. The long-term destiny of transplanted stem cells in vivo. Fig.S2. Dio-labeled ApoEVs successfully loaded onto Gel/ECM scaffold at different magnification. Fig.S3. Vesicle structure loaded onto the scaffold under scanning electron microscope. Fig.S4. Results of Young's modulus of three groups of scaffolds. Table S1. Upregulated miRNA-related functions in H-ApoEVs. Table S2. The sequences of primer for the RT-qPCR. Table S3. International Cartilage Repair Society (ICRS) macroscopic evaluation guidelines. Table S4. Modified O’Driscoll score system

Table_1_Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial – the safe iron study.DOCX

IntroductionThe safety of novel forms of iron in healthy, iron-replete adults as might occur if used in population-based iron supplementation programs was examined. We tested the hypotheses that supplementation with nanoparticulate iron hydroxide adipate tartrate (IHAT), an iron-enriched Aspergillus oryzae product (ASP), or ferrous sulphate heptahydrate (FS) are safe as indicated by erythrocyte susceptibility to malarial infection, bacterial proliferation, and gut inflammation. Responses to FS administered daily or weekly, and with or without other micronutrients were compared.MethodsTwo phases of randomized, double-blinded trials were conducted in Boston, MA. Phase I randomized 160 volunteers to six treatments: placebo, IHAT, ASP, FS, and FS plus a micronutrient powder (MNP) administrated daily at 60 mg Fe/day; and FS administered as a single weekly dose of 420 mg Fe. Phase II randomized 86 volunteers to IHAT, ASP, or FS administered at 120 mg Fe/day. Completing these phases were 151 and 77 participants, respectively. The study was powered to detect effects on primary endpoints: susceptibility of participant erythrocytes to infection by Plasmodium falciparum, the proliferation potential of selected pathogenic bacteria in sera, and markers of gut inflammation. Secondary endpoints for which the study was not powered included indicators of iron status and gastrointestinal symptoms.ResultsSupplementation with any form of iron did not affect any primary endpoint. In Phase I, the frequency of gastrointestinal symptoms associated with FS was unaffected by dosing with MNP or weekly administration; but participants taking IHAT more frequently reported abdominal pain (27%, p DiscussionWith respect to the primary endpoints, few differences were found when comparing these forms of iron, indicating that 28 days of 60 or 120 mg/day of IHAT, ASP, or FS may be safe for healthy, iron-replete adults. With respect to other endpoints, subjects receiving IHAT more frequently reported abdominal pain and nausea, suggesting the need for further study.Clinical Trial RegistrationClinicalTrials.gov, NCT03212677; registered: 11 July 2017.</p