Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Activity Assay, Expressing, Functional Assay, Quantitative RT-PCR, esiRNA

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Negative Control, Quantitative RT-PCR, IF-P, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Representative immunostaining of LV sections from control Cre mice. Serial sections of LVs from control Cre mice at 8 days post-MI were stained with anti–Mac-2 or anti-GATA3 antibodies. GATA3-positive staining is clearly visible in the infarcted region of the myocardium, which contains large numbers of macrophages, but not in the healthy region. Sections were also stained with fluorescent-labeled antibodies to demonstrate the nuclear colocalization of GATA3 in macrophages located in the infarcted region of the myocardium. (B) Representative M-mode echocardiograms from the 2 groups of mice before (BL) and 1 month after MI (1M). (C) Representative photographs of Masson Trichrome staining from sections from the base to the apex of hearts from the 2 mice genotype groups. Photographs of all of the hearts are shown in Online Figure 3. (D) Morphometric analysis of each heart section. The number of animals in each group is shown in Table 1. LV = left ventricle; mGATA3KO = myeloid-specific GATA3 knockout mouse; MI = myocardial infarction.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Immunostaining, Control, Staining, Labeling, Knock-Out

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Representative M-mode echocardiographic data from the 2 mouse genotypes at 2 months after transverse aortic constriction. (B) Harvested hearts were analyzed to determine the size of cardiomyocytes and collagen content. N = 5 mice/genotype. The number of animals in each group is shown in Table 2. mGATA3KO = myeloid-specific GATA3 knockout mouse.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Knock-Out

Journal: Journal of the American College of Cardiology

Article Title: Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy

doi: 10.1016/j.jacc.2018.05.061

Figure Lengend Snippet: (A) Macrophages were isolated from the peritoneum or the bone marrow of control Cre mice and cultured. Cultured cells were treated with IL-4 at the indicated times, and GATA3 expression was evaluated by qPCR. GATA3 gene expression data were normalized against GAPDH. Results are the mean of triplicate determinations ± SD. (B) Proteins were extracted from cells treated with 20 ng/ml of IL-4 at the indicated times. (C) Dose-response analysis of GATA3 expression was determined by qPCR using cultured macrophages isolated from the bone marrow of control mice. (D) Proteins were analyzed by western blot analysis of lysates from cells treated with 10 or 20 ng/ml of IL-4. (E) Bone marrow-derived macrophages were plated onto chamber slides and treated with 20 ng/ml IL-4 for 4 h, followed by staining with the indicated fluorescent antibodies. Isotypematched antibodies were used as controls. (F) Total RNA was isolated from cultured bone marrow-derived macrophages from Cre mice treated with IL-4 at the indicated times and analyzed by qPCR using primers specific to the proximal (E1b) or distal (E1a) GATA3 promoter. GATA3 gene expression data were normalized against the GAPDH gene. (G) Cultured bone marrow-derived macrophages were treated with the indicated agonists for 2 and 24 h. Total RNA was extracted and analyzed by qPCR, and the resulting GATA3 data were normalized against GAPDH. (H) Cultured bone marrow-derived macrophages from the indicated mouse genotype were treated with IL-4 or IFNγ for 2 h, followed by isolation of total RNA. qPCR data for Arg-1 expression were normalized against GAPDH gene expression. In addition, proteins were extracted from the treated cells and analyzed by western blot using an antibody to Arg-1. All qPCR results are means of triplicate determinations ± SD. *Indicates a significant difference between control and treated cells, p < 0.05. (I) Representative immunostaining data for IL-4 or IL-33 expression in LV sections from control mice at 8 days post-MI. (J) Representative immunostaining of the LV section from the mGATA3KO mice stained with antibodies to Mac-2, IL-4, or IL-33. The upper panels show low magnification and the lower panels show high magnification. Arg-1 = arginase-1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase IFN = interferon; IL = interleukin; qPCR = quantitative real-time polymerase chain reaction. Other abbreviations as in Figure 1.

Article Snippet: The anti-GATA3 antibody (clone D-16; sc-22206, Santa Cruz Biotechnology, Dallas, Texas) was used for GATA3 staining.

Techniques: Isolation, Control, Cell Culture, Expressing, Gene Expression, Western Blot, Derivative Assay, Staining, Immunostaining, Real-time Polymerase Chain Reaction