Journal: Cell Death & Disease

Article Title: Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2

doi: 10.1038/s41419-019-1742-7

Figure Lengend Snippet: a Western blotting analysis showing that Neat1 knockdown enhanced P21 protein expression but decreased Cdk4 protein expression in C2C12 cells. The relative protein levels of P21 and Cdk4 were quantified using ImageJ software. b , c ChIP-qPCR results revealed that the enrichments of Ezh2 ( b ) and H3k27me3 ( c ) at the P21 promoter were significantly decreased after Neat1 knockdown. d , e ChIP-qPCR results revealed that the enrichments of Ezh2 ( d ) and H3k27me3 ( e ) at the P21 promoter were significantly increased after Neat1 overexpression. f Co-transfection of Ezh2 siRNA fragment and Neat1 expression vector in C2C12 cells for 2 days. Immunofluorescence staining of P21 was performed, and P21 expression was quantified by ImageJ. The quantification of P21 immunofluorescence staining results showed that the overexpression of Neat1 inhibited P21 protein expression, but had no significant effect on P21 expression after co-transfection with Ezh2 siRNA fragment. g Neat1 siRNA fragment and Ezh2 expression vector were co-transfected into C2C12 cells and the cells were performed with EdU staining at 2 days after transfection. The percentage of EdU + cells was quantified. The quantification of EdU staining results showed that Neat1 knockdown inhibited myoblast proliferation. After co-transfected with Ezh2 expression vector, Neat1 knockdown can not inhibit myoblast proliferation. h Neat1 and P21 siRNA fragments were co-transfected into C2C12 cells and the cells were performed with EdU staining at 2 days after transfection. The quantification of EdU staining results showed that Neat1 knockdown significantly reduced the percentage of EdU + cells, but did not reduce the number of EdU + cells after co-transfection with P21 siRNA fragment. Protein levels were normalized to those of β-actin. All values represent the mean ± s.d. of three independent experiments. * p < 0.05, ** p < 0.01, N.S. indicates not significant

Article Snippet: The antibodies and their dilutions were shown as following: Myod (Santa Cruz Biotechnology, USA; sc-760; 1:1000), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:200), Myhc (Santa Cruz Biotechnology, USA; sc-376157; 1:3000), α-actin (Proteintech, China; 23660-1-AP; 1:1000), Tnni2 (Abcam, UK; ab184554; 1:1000), P21 (BOSTER, China; BM4382; 1:200), Ezh2 (Cell Signaling Technology, USA; 5246; 1:1000), Pcna (Servicebio, China; GB11010; 1:500), Ki67 (Abcam, UK; ab16667; 1:1000), β-actin (Santa Cruz Biotechnology, USA; sc-4777; 1:1000), Gapdh (BOSTER, China; BM3876; 1:200), Pax7 (Developmental Studies Hybridoma Bank; USA; 1:1000), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:1000).

Techniques: Western Blot, Knockdown, Expressing, Software, ChIP-qPCR, Over Expression, Cotransfection, Plasmid Preparation, Immunofluorescence, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2

doi: 10.1038/s41419-019-1742-7

Figure Lengend Snippet: In proliferating myoblasts, Neat1 guides Ezh2 to the P21 promoter and inhibits P21 expression, leading to the promotion of myoblast proliferation. Upon differentiation, Neat1 recruits Ezh2 to inhibit the expression of muscle-specific genes, such as Myog , Myh4 , and Tnni2 , and suppresses myogenic differentiation

Article Snippet: The antibodies and their dilutions were shown as following: Myod (Santa Cruz Biotechnology, USA; sc-760; 1:1000), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:200), Myhc (Santa Cruz Biotechnology, USA; sc-376157; 1:3000), α-actin (Proteintech, China; 23660-1-AP; 1:1000), Tnni2 (Abcam, UK; ab184554; 1:1000), P21 (BOSTER, China; BM4382; 1:200), Ezh2 (Cell Signaling Technology, USA; 5246; 1:1000), Pcna (Servicebio, China; GB11010; 1:500), Ki67 (Abcam, UK; ab16667; 1:1000), β-actin (Santa Cruz Biotechnology, USA; sc-4777; 1:1000), Gapdh (BOSTER, China; BM3876; 1:200), Pax7 (Developmental Studies Hybridoma Bank; USA; 1:1000), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:1000).

Techniques: Expressing

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Annotation of PSPC1 ChIP-seq peaks in ESCs at promoters, intergenic or genic regions. (B) Mean intensity plot by reads per million (RPM) showing PSPC1 ChIP-seq intensity of WT and Pspc1 KO ESCs at gene bodies (within 3K bp). TSS, transcription start site, TTS, transcription termination site. (C) Overlap ofthe PSPC1, PRC2 subunit SUZ12, and TET1 peaks in ESCs. (D) Mean intensity plot by RPM showing PSPC1, TET1, and RPC2 subunit SUZ12 ChIP-seq intensity at PSPC1 peak regions (within 5K bp at PSPC1 peak center). (E) Heatmaps by RPM showing PSPC1 and histone marks H3K4me3, H3K27ac , and H3K27me3 at PSPC1/TET1 common peak regions (within 8K bp at PSPC1 peak center) with and without PRC2 occupancy. (F) ChIP-seq tracks of PSPC1, TET1, SUZ12, and histone marks of H3K4me3, H3K27me3, and H3K27ac at PSPC1/TET1 common peak regions with ( T , Fgf5 ) and without ( Pou5f1 , Nanog ) PRC2 occupancy. The numbers indicate the normalized RPM value of the tracks.

Article Snippet: Immunoprecipitation was performed with the following primary antibodies: PSPC1 (Santa Cruz, sc-84577 and Bethyl, A303-206A), SUZ12 (Active Motif, 39357), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Cell Signaling, 9733S), TET1 (GenTex, GTX125888), or rabbit IgG (Millipore, PP64) overnight at 4°C with continuous mixing, followed by incubation with protein G dynaberads (Invitrogen, 10004D) for another 2 h at 4°C.

Techniques: ChIP-sequencing

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Schematic depiction of the Neat1 KO strategy. The scissors denote two gRNA-targeting sites for CRISPR-Cas9 genome editing. The short ( Neat1_1 ) and long ( Neat1_2 ) isoforms of the mouse Neat1 gene are indicated. (B) RNA-seq tracks (left) and expression of Neat1 (right) during the ESC-to-EpiLC differentiation. The numbers indicate the normalized RPM value of the tracks (left). Neat1 expression is shown in FPKM (fragments per kilobase of transcript per million mapped reads) values (right). Error bars represent the standard deviation of biological duplicates. (C) Scatter plot of the relative gene expression of DEGs upon Pspc1 KO or Neat1 KO relative to WT at D2 EpiLC from RNA-seq analysis; p value is from the Fisher exact test. Representative genes are labeled on the plot. (D) RNA-seq tracks of WT and Neat1 KO ESCs and EpiLCs at bivalent gene loci ( Fgf5 and Nefl ). The numbers indicate the normalized RPM value of the tracks. (E) RT-qPCR analysis of bivalent genes in WT and Neat1 KO ESCs (46C genetic background with two independent clones, 5F and 7G) during EpiLC differentiation. Error bars represent the standard deviation of technical triplicates. (F) Schematic depiction of Neat1 ChIRP-seq analysis in WT and Neat1 KO ESCs and D2 EpiLCs. Biotinylated probes based on their relative positions along the Neat1_1 RNA were ranked and split into odd and even probes, followed by streptavidin pull-down and DNA sequencing. (G) Mean intensity plot by RPM showing Neat1 ChIRP-seq intensity enriched at the Neat1 peak regions in ESCs and D2 EpiLCs (within 1K bp around Neat1 peak regions identified in ESCs). (H) Boxplots depicting quantification of Neat1 ChIRP-seq intensity by RPM at PSPC1 ChIP-seq peak regions (extend 5K bp, identified in ESCs) from WT and Neat1 KO ESCs and D2 EpiLCs. p value is from the Mann-Whitney test. (I) PSPC1, TET1, and SUZ12 ChIP-seq tracks in ESCs and Neat1 ChIRP-seq tracks in WT and Neat1 KO ESCs and D2 EpiLCs at the promoters of bivalent genes ( T , Fgf8, Sp8, and Wnt3 ). The numbers indicate the normalized RPM value of the tracks.

Article Snippet: Immunoprecipitation was performed with the following primary antibodies: PSPC1 (Santa Cruz, sc-84577 and Bethyl, A303-206A), SUZ12 (Active Motif, 39357), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Cell Signaling, 9733S), TET1 (GenTex, GTX125888), or rabbit IgG (Millipore, PP64) overnight at 4°C with continuous mixing, followed by incubation with protein G dynaberads (Invitrogen, 10004D) for another 2 h at 4°C.

Techniques: CRISPR, RNA Sequencing, Expressing, Standard Deviation, Gene Expression, Labeling, Quantitative RT-PCR, Clone Assay, DNA Sequencing, ChIP-sequencing, MANN-WHITNEY

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Schematic depiction of the Tet1 -degron knock-in (KI) strategy using CRISPR-Cas9 genome-editing tool (the scissor symbol). The HA-tagged FKBP12 F36V donor sequence is inserted right after the start codon (ATG) of TET1 CDS to create the in-frame fusion protein. (B) Western blot analysis of TET1 protein in Tet1 -degron ESCs (two independent clones, C#13 and C#16) upon dTAG13 treatment for 24 h. Degradation of TET1 was indicated by both endogenous antibody and HA fusion protein tag. (C) Schematic depiction of the bivalent histone marks H3K4me3 and H3K27me3 and the PRC2 subunit SUZ12 ChIP-seq analysis in ESCs and D2 EpiLCs of different genotypes ( Pspc1 WT/KO and Neat1 WT/KO) or treatment ( Tet1 -degron with control/dTAG13). (D) Overlap of the bivalent peaks (H3K4me3 and H3K27me3) identified in ESCs and EpiLCs and with the PSPC1/TET1/SUZ12 common peaks identified in ESCs. (E) Mean intensity plot (top) and heatmap (bottom) by RPM of SUZ12 ChIP-seq intensity in WT ESCs and EpiLCs at SUZ12 peak regions (within 5K bp at peak center, identified in ESCs). (F and G) Mean intensity plot (top) and heatmap (bottom) by RPM of SUZ12 (F) and H3K27me3 (G) ChIP-seq intensity in D2 EpiLCs at PSPC1/TET1/SUZ12 common peak regions (within 5K bp at peak center, identified in ESCs). (H) SUZ12, H3K4me3, and H3K27me3 ChIP-seq tracks at the promoters of bivalent genes ( Fgf5 , Nefl, Sall2, Eomes, and Wnt3 ) in D2 EpiLCs. The numbers indicate the normalized RPM value of the tracks.

Article Snippet: Immunoprecipitation was performed with the following primary antibodies: PSPC1 (Santa Cruz, sc-84577 and Bethyl, A303-206A), SUZ12 (Active Motif, 39357), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Cell Signaling, 9733S), TET1 (GenTex, GTX125888), or rabbit IgG (Millipore, PP64) overnight at 4°C with continuous mixing, followed by incubation with protein G dynaberads (Invitrogen, 10004D) for another 2 h at 4°C.

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Clone Assay, ChIP-sequencing, Control

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Co-IP of PSPC1 and SUZ12 in ESCs using a nucleosome-containing protocol (see for detail). (B) Biotinylated Neat1 (bio Neat1 ) RNAs pull down both EZH2 and PSPC1. Left: streptavidin (SA) beads conjugated with Neat1 sense (S) or antisense (AS) RNA, and empty beads(EB) were used for pull-down from ESC nuclear lysates followed by western blot analysis of bio Neat1 -bound proteins. EZH2 and PSPC1 blots of both short and long exposure (exp.) are shown. Right: bio Neat1 sense (S) or antisense (AS) RNA were transcribed by in vitro transcription (IVT) and confirmed by SA-HRP dot blot. ESC total RNA serves as a negative control. (C) EZH2 and PSPC1 CLIP-qPCR analysis of Neat1 in WT, Pspc1 KO, and Ezh2 KO ESCs. Gapdh serves as a negative control; p value is from two-tailed t test, and “n.s.” denotes statistically non-significant. (D) Mean intensity plot (top) and heatmap (bottom) by RPM of Neat1 ChIP-seq intensity at the bivalent regions (within 5K bp at peak center, identified in ESCs) in ESCs and D2 EpiLCs. (E) EZH2 CLIP-qPCR analysis of bivalent gene mRNAs ( Fgf5, Nefl , and Sall2 ) in D2 EpiLCs of different genotypes (WT versus KO); p value is from the two-tailed t test. (F) PSPC1 CLIP-qPCR analysis of Neat1 and bivalent genes’ transcripts ( Fgf5 , Neff , and Sall2 ) in D2 EpiLCs of different genotypes. Error bars in (C), (E), and (F) represent the standard deviation of technical triplicates. Experiments were repeated in biological duplicates.

Article Snippet: Immunoprecipitation was performed with the following primary antibodies: PSPC1 (Santa Cruz, sc-84577 and Bethyl, A303-206A), SUZ12 (Active Motif, 39357), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Cell Signaling, 9733S), TET1 (GenTex, GTX125888), or rabbit IgG (Millipore, PP64) overnight at 4°C with continuous mixing, followed by incubation with protein G dynaberads (Invitrogen, 10004D) for another 2 h at 4°C.

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Dot Blot, Negative Control, Two Tailed Test, ChIP-sequencing, Standard Deviation

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunoprecipitation was performed with the following primary antibodies: PSPC1 (Santa Cruz, sc-84577 and Bethyl, A303-206A), SUZ12 (Active Motif, 39357), H3K4me3 (EpiCypher, 13-0041), H3K27me3 (Cell Signaling, 9733S), TET1 (GenTex, GTX125888), or rabbit IgG (Millipore, PP64) overnight at 4°C with continuous mixing, followed by incubation with protein G dynaberads (Invitrogen, 10004D) for another 2 h at 4°C.

Techniques: Recombinant, Affinity Purification, Mass Spectrometry, Methylated DNA Immunoprecipitation Sequencing, Software

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A) MC3T3 cells were differentiated to mineralized osteoblasts for 9 days of culture (DIV) using Ascorbic Acid (AA) 50ug/mL. Nuclear extracts were collected at indicated the days and analyzed by western blot using specific antibodies against the indicated epigenetic regulators. TFIIB or RNA-polymerase II (RNAPII) was used to control for equal protein loading. (B-C and E-F) Binding of chromatin regulators to the Runx2 P1 promoter at the indicated differentiation days were analyzed by ChIP using antibodies against: (B) Wdr5, (C) Utx, (E) Ezh2, (F) Prmt5, and (G) Jarid1b. (D) Re-ChIP assay performed in chromatin samples obtained from differentiated cells (5 DIV) using first an antibody against Utx and subsequently an antibody against Wdr5. Results and statistical analyses are shown as described in figure legend 2. ***p<0.001, *p<0.05, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Western Blot, Binding Assay

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A-B and D-E) Differentiating MC3T3 cells (3DIV) were infected with lentiviral particles containing shRNAs against the chromatin modifiers: (A) Wdr5, (B) Utx, (D) Ezh2 and (E) Prmt5. Knockdown efficiencies were confirmed by RT-qPCR and western blot (A-D, left panels) analyses, 48 h post-infection (5 DIV). TFIIB or RNAPII proteins were used to control for equal protein loading. (C) Effect of knocking down Wdr5 (left) and Utx (right) expression on Bglap gene transcription. mRNA expression values were normalized against Gapdh mRNA levels. Statistical analyses were assessed with respect to the mRNA levels obtained in cells infected with an empty vector (EV). *p<0.05, ***p<0.001, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation