MicroRNA-181 Regulates CARM1 and Histone Aginine Methylation to Promote Differentiation of Human Embryonic Stem Cells

<div><p>As a novel epigenetic mechanism, histone H3 methylation at R17 and R26, which is mainly catalyzed by coactivator-associated protein arginine methyltransferase 1 (CARM1), has been reported to modulate the transcription of key pluripotency factors and to regulate pluripotency in mouse embryos and mouse embryonic stem cells (mESCs) in previous studies. However, the role of CARM1 in human embryonic stem cells (hESCs) and the regulatory mechanism that controls <em>CARM1</em> expression during ESCs differentiation are presently unknown. Here, we demonstrate that CARM1 plays an active role in the resistance to differentiation in hESCs by regulating pluripotency genes in response to BMP4. In a functional screen, we identified the miR-181 family as a regulator of <em>CARM1</em> that is induced during ESC differentiation and show that endogenous miR-181c represses the expression of <em>CARM1</em>. Depletion of CARM1 or enforced expression of miR-181c inhibits the expression of pluripotency genes and induces differentiation independent of BMP4, whereas overexpression of <em>CARM1</em> or miR-181c inhibitor elevates Nanog and impedes differentiation. Furthermore, expression of <em>CARM1</em> rescue constructs inhibits the effect of miR-181c overexpression in promoting differentiation. Taken together, our findings demonstrate the importance of a miR-181c-CARM1 pathway in regulating the differentiation of hESCs.</p> </div

miR-181c leads to hESC differentiation through negative regulation of <i>CARM1</i> and H3R17 methylation.

<p>(A) Overexpression of miR-181c down-regulated <i>CARM1</i> expression in comparison to negative control (NC) RNA-transfected ESCs at both the mRNA and protein levels, as determined by qRT-PCR and Western blotting, respectively. H3R17 methylation level and <i>Oct4</i>, <i>Nanog</i> and <i>Sox2</i> mRNA expression were also monitored. Samples were assayed in duplicate (n = 3) and normalized to endogenous <i>β-actin</i> expression. **, p<0.01. (B) Expression of a subset of differentiation genes in ESCs that were transfected with miR-181c or NC RNA or were not transfected was monitored by qRT-PCR. Mean levels (after 3 days) of expression are shown relative to the NC RNA-transfected sample (shown as one fold) and normalized to <i>β-actin</i> expression levels. **, p<0.01. (C, D, E) miR-181c mimics were transfected into ESCs with the 3′UTR-deficient-<i>CARM1</i>-expressing plasmid (<i>CARM1</i> OE) or the control plasmid (pcDNA3). ESCs co-transfected with NC RNA and pcDNA3 were used as controls. (C) Pluripotency was examined by AP staining 8 days after transfection. The images for the whole plate or representative clones are shown. Scale bar: 500 µm. CARM1 protein expression is also shown. **, p<0.01. (D) Expression of a subset of differentiation genes in ESCs transfected with miR-181c+pcDNA3 or miR-181c+<i>CARM1</i> or NC RNA was monitored by qRT-PCR. Mean levels (after 3 days) of expression are shown relative to the NC RNA-transfected sample (shown as one fold) and normalized to <i>β-actin</i> expression levels. (E) <i>Oct4</i>, <i>Nanog</i> and <i>Sox2</i> mRNA expression was also monitored by qRT-PCR. Samples were assayed in duplicate (n = 3) and normalized to endogenous <i>β-actin</i> expression. **, p<0.01. (F) ChIP analysis of hESCs overexpressing <i>CARM1</i> or miR-181c. ChIP was performed on sonicated chromatin using anti-CARM1 or anti-H3R17-di-me antibodies. Immunoprecipitated DNA was analyzed by qRT-PCR with primers targeting the promoter regions of the investigated gene. The fold enrichment value is shown as the normalized ChIP signal divided by the normalized input signal. Cells transfected with NC RNA or pcDNA3 were used as negative controls. The results of the electrophoretic analysis are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053146#pone.0053146.s003" target="_blank">Fig. S3A</a>. *, p<0.05; **, p<0.01.</p

The miR-181 family directly regulates <i>CARM1</i> expression in hESC.

<p>(A) The expression levels of mature miRNAs predicted to target the <i>CARM1</i> 3′UTR were monitored in differentiated ESCs by qRT-PCR and normalized to endogenous <i>U6</i> expression. *, p<0.05; **, p<0.01. (B) The chromosome positions of the primary transcripts of miR-181 family members are shown, their expression levels in differentiated ESCs were determined by qRT-PCR and normalized to endogenous <i>β-actin</i> expression. **, p<0.01. (C) The predicted consequential pairing of miRNAs and their target regions in the wild-type <i>CARM1</i> 3′UTR or the mutant (mut) <i>CARM1</i> 3′UTR are shown. (D) The HEK293 cells were co-transfected with the firefly-luciferase-expressing vector pMIR-REPORT containing the wild-type <i>CARM1</i> 3′UTR, the mut <i>CARM1</i> 3′UTR or the control insert as well as the internal control renilla-luciferase-expressing vector pRL-TK and the indicated RNAs. After 48 h, the luciferase activities were measured. The data were normalized by dividing firefly luciferase activity with that of Renilla luciferase, **, p<0.01. (D) Luciferase activities were measured in human ESCs co-transfected with the pMIR-REPORT plasmid containing wild-type <i>CARM1</i> 3′UTR, mut <i>CARM1</i> 3′UTR or control insert and the internal control, pRL-TK. All the samples were assayed in duplicate (n = 3). **, p<0.01.</p

The miR-181c inhibitor suppresses hESC differentiation.

<p>(A) Enforced expression of the miR-181c inhibitor down-regulated mature miR-181c levels relative to negative control (NC) RNA-transfected ESCs, as shown by qRT-PCR. **, p<0.01. Western blotting detected that CARM1 and H3R17me2 protein levels were clearly decreased. (B, C, D) The effect of miR-181c inhibition on differentiated hESCs. Human ESCs were transfected with miR-181c inhibitor or NC RNA and then induced to differentiate by the addition of BMP4 in the absence of bFGF. (B) The expression of <i>CARM1</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Oct4</i> at the mRNA level was quantified by qRT-PCR, CARM1 and Nanog protein expression levels were also quantified by Western blotting. (C) Pluripotency was examined by AP staining 3 days after transfection. The counts of AP-positive clones and the images of the representative plates are shown. Samples were assayed in duplicate (n = 3). **, p<0.01. (D) Expression of a subset of differentiation-associated genes in ESCs transfected with miR-181c inhibitor or NC RNA were monitored by qRT-PCR and normalized to <i>β-actin</i> expression levels. Mean levels (after 8 days) expressed relative to undifferentiated hESCs (shown as one fold) are shown. (E) Model for the miR-181/CARM1/core-pluripotency-factors regulatory loop in the modulation of hESC pluripotency. Pluripotency is maintained in ESCs in part by histone H3 arginine methylation by CARM1 at the <i>Oct4</i>, <i>Nanog</i> and <i>Sox2</i> promoters. The core pluripotency factors also recruit H3K27 methylases to the miR-181c promoter to inhibit its expression. In differentiated hESCs, H3K27 methylation is inhibited due to the reduction of core pluripotency factors, and miR-181 family members are subsequently induced and down-regulate CARM1 activity. H3R17me2 production is eventually stopped, which aggravates the decrease in expression of core pluripotency factors as well as the loss of pluripotency.</p

CARM1 is down-regulated post-transcriptionally during hESC differentiation.

<p>(A, B) The kinetic expression levels of <i>CARM1</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Oct4</i> in differentiated hESCs. Human ESCs differentiation was induced by the addition of BMP4 in the absence of bFGF. The expression levels of <i>CARM1</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Oct4</i> at the mRNA (A) and protein levels (B) were quantified by qRT-PCR and Western blotting, respectively. *, p<0.05; **, p<0.01. (C, D) Overexpression of 3′UTR-deficient <i>CARM1</i> rescued the down-regulation of key pluripotency factors during hESC differentiation. Differentiation of hESCs overexpressing 3′UTR-deficient <i>CARM1</i> was induced, and the expression levels of <i>CARM1</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Oct4</i> at the mRNA (C) and protein levels (D) were quantified by qRT-PCR and Western blotting, respectively. Blank pcDNA3 was used as a negative control. Samples were assayed in duplicate (n = 3) and normalized to endogenous <i>β-actin</i> expression. **, p<0.01. (E) Knockdown of <i>CARM1</i> in ESCs using siRNAs resulted in the down-regulation of <i>CARM1</i>, <i>Nanog, Sox2</i> and <i>Oct4</i> mRNA levels. <i>CARM1</i>-specific siRNAs were transfected every 4 days; total RNA was extracted from hESCs 8 days after the first transfection and quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Mean values of the indicated transcript levels are plotted as percentages relative to those after transfection with negative control RNA (NC RNA). Samples were assayed in duplicate (n = 3) and normalized to endogenous <i>β-actin</i> expression. **, p<0.01. (F) The pluripotency of hESCs was examined by AP staining 8 days after siRNA transfection. NC RNA-transfected ESCs were used as controls. The counts of AP-positive clones per field were quantified in duplicate (n = 3), and the images for the representative plates are all shown. **, p<0.01.</p

Multi_CycGT: A Deep Learning-Based Multimodal Model for Predicting the Membrane Permeability of Cyclic Peptides

Cyclic peptides are gaining attention for their strong binding affinity, low toxicity, and ability to target “undruggable” proteins; however, their therapeutic potential against intracellular targets is constrained by their limited membrane permeability, and researchers need much time and money to test this property in the laboratory. Herein, we propose an innovative multimodal model called Multi_CycGT, which combines a graph convolutional network (GCN) and a transformer to extract one- and two-dimensional features for predicting cyclic peptide permeability. The extensive benchmarking experiments show that our Multi_CycGT model can attain state-of-the-art performance, with an average accuracy of 0.8206 and an area under the curve of 0.8650, and demonstrates satisfactory generalization ability on several external data sets. To the best of our knowledge, it is the first deep learning-based attempt to predict the membrane permeability of cyclic peptides, which is beneficial in accelerating the design of cyclic peptide active drugs in medicinal chemistry and chemical biology applications

Additional file 1: of Transcriptomic analysis of a moderately growing subisolate Botryococcus braunii 779 (Chlorophyta) in response to nitrogen deprivation

Table S1. List of the 12041 annotated ESTs in B. braunii 779

The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

<div><p>Transcription termination in <i>Saccharomyces cerevisiae</i> can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) <i>via</i> the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of <i>ctk1</i>Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a <i>CTK1</i> deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.</p></div

Partial suppression of <i>ctk1Δ</i> termination phenotype by a <i>spt5</i>Δ<i>CTR</i> mutant.

<p>Northern blot showing the effect of the <i>spt5</i>Δ<i>CTR</i> mutation on termination at <i>snR47</i> and the <i>NEL025c</i> CUT in strains metabolically depleted for Ctk1p and Bur1p. <i>CTK1</i> and <i>BUR1</i> were expressed under control of the <i>GAL1</i> promoter and depletion was carried for the times indicated. Note the appearance of longer RNA species upon depletion of <i>CTK1</i> that are suppressed in an <i>spt5</i>Δ<i>CTR</i> background. An <i>rrp6Δ</i> background was used to allow detection of unstable RNA species. Transcripts were revealed with double stranded probes that span the entire <i>snR47</i>, <i>NEL025c</i> genes or the 3′ end of <i>ACT1</i>.</p

Recruitment of Nrd1p and Pcf11p, relative to Rpb3p.

<p>(A, B, and C) Scheme of the genomic regions analyzed by ChIP-qPCR. (D, E, and F) Analysis of Nrd1p occupancy relative to Rpb3p occupancy as determined by ChIP in wild type and <i>ctk1</i>Δ strains at the <i>snR5</i>, <i>snR13</i> and <i>snR82</i> loci. Relative ChIP values are normalized to the TEL-V signal in wt. (G, H, and I) Analysis of Pcf11p occupancy relative to Rpb3p occupancy in wt and <i>ctk1</i>Δ at <i>snR5</i>, <i>snR13</i> and <i>snR82</i>. Relative ChIP values are normalized to the TEL-V signal in wt. Error bars represent standard errors. Raw Rpb3p, Nrd1p and Pcf11p levels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080495#pone.0080495.s005" target="_blank">Figure S5</a>.</p