Journal: bioRxiv

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

doi: 10.1101/2021.04.21.440535

Figure Lengend Snippet: ( a ) Illustration of experimental approach tracing the effect of developmental loss of subunit POLR3G, subunit-specific disruption of POLR3G, and cancer-associated re-establishment of POLR3G and accompanying chromatin features, which collectively identify POLR3G-driven modulation of Pol III transcription potential in proliferating cells. ( b ) Pol III subunit and transcription factor legend corresponding to genome-wide maps in panels c-k, including POLR3A/RPC1 (3A); POLR3B/RPC2 (3B); POLR1D/RPAC2 (1D); POLR3C/RPC3 (3C); POLR3G/RPC7α (3G); POLR3GL/RPC7β (3GL); POLR3D/RPC4 (3D); POLR3E/RPC5 (3E); BRF1/TFIIIB90; TF3C1/TFIIIC220. Corresponding subcomplex indicated. ( c-k ) Example ChIP-seq read signals for each subunit/factor are shown in THP-1 monocytes across canonical Pol III-transcribed genes of varying promoter architecture, including ribosomal 5S rRNA genes (type 1 promoter; panel c ), tRNA, 7SL, vault, and SNAR RNA genes (type 2 promoter, panels d-g , respectively), and Y, U6, 7SK, and RMRP RNA genes (type 3 promoter, panels h-k , respectively). Gene labels include Unique RNA Sequence (URS) identifiers assigned by (and connected to) the RNAcentral database. Bottom panel illustrates corresponding promoter architecture classification. ( l ) Cartoon schematic of the Pol III protein complex with emphasis on subunits mapped in this study (labeled) and the corresponding color code for genomic signal plots. Illustration serves as general reference guide inspired by previous structural reconstructions (Hoffmann et al., Nature 2015) . ( m ) Scatterplot visualization of the correlation between normalized read densities for paralogous Pol III subunits, POLR3G and POLR3GL, at Pol III complex-occupied genes in THP-1 monocytes.

Article Snippet: 5 ug of antibody per ChIP was coupled to 18 uL of beads and rotated overnight with sheared chromatin at 4° C. Beads were then washed 5x in ChIP wash buffer (Santa Cruz), 1x in TE, and chromatin eluted in TE + 1% SDS.

Techniques: Disruption, Genome Wide, ChIP-sequencing, Sequencing, Labeling

Journal: bioRxiv

Article Title: A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

doi: 10.1101/2021.04.21.440535

Figure Lengend Snippet: ( a ) Heatmap visualization of ChIP-seq signal density over individual Pol III-transcribed genes for Pol III subunits POLR3A, POLR3B, POLR1D, POLR3C, POLR3G, POLR3GL, POLR3D, and POLR3E, TFIIIB subunit BRF1, and TFIIIC subunit TF3C1. Heatmap is ordered by the median signal density across Pol III subunits over canonical Pol III-transcribed genes (n = top 1,000 canonical genes; RNAcentral annotation). Corresponding gene type indicated by right-flanking colorbar. ( b ) Heatmap visualization of Pearson correlation between chromatin IP experiments for individual Pol III subunits and BRF1, GTF3C1. ( c-h ) Correlation scatterplot visualization between Pol III subunit POLR3G and Pol III subunits POLR3A, POLR3B, POLR1D, POLR3C, POLR3D, and POLR3E, respectively. ( i ) Scatterplot visualization of correlation between Pol III subunit POLR3G signal enrichment and chromatin accessibility profile (ATAC-seq) at Pol III occupied genes in THP-1 monocytes. ( j ) Analogous correlation plot between chromatin accessibility profile (ATAC-seq) and median Pol III subunit signal enrichment. ( k ) Gene expression profile for mapped Pol III subunits in THP-1 cells (points represent individual biological replicates).

Article Snippet: 5 ug of antibody per ChIP was coupled to 18 uL of beads and rotated overnight with sheared chromatin at 4° C. Beads were then washed 5x in ChIP wash buffer (Santa Cruz), 1x in TE, and chromatin eluted in TE + 1% SDS.

Techniques: ChIP-sequencing, Chromatin Immunoprecipitation, Gene Expression

Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Valproic acid-enhanced cardiac differentiation. (A) P19 stem cells were treated with DMSO or increasing concentrations of valproic acid (VPA 0.5, 1, 2 mM) during EB formation, maintained in the tissue culture dishes for 3 additional days without treatments, and then stained for myosin heavy chain and cTnT. Quantification is presented as fractions of cells differentiated into cardiomyocytes relative to the total cell populations. Error bars represent the standard deviations of three independent experiments. (B) Shown are the representative microscopy images of the cells stained for cTnT (green). Hoechst was used to stain the DNA (blue) concomitantly (scale bars = 50 μm). (C) Western analysis of GATA4 protein expression and the levels of global H3 acetylation. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were included as a negative control. Shown are the cropped blot images representing indicated protein. (D) Occupancy of p300 at the GATA4 promoter (GATApro) and a control locus (GATActl) were examined by a real-time PCR based ChIP analysis. Quantification is presented as the fold variations of undifferentiated control.

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Staining, Microscopy, Western Blot, Expressing, Negative Control, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Occupancy of p300 at the GATA4 promoter at early stage of differentiation. (A) P19 cells were differentiated with DMSO and co-treatment of curcumin (10 μM) was during the first 2 days of EB formation. The cellular levels of H3 acetylation and p300 protein were analyzed by Western blotting on day 4. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were used as the negative control. Shown are the cropped blot images representing indicated protein. (B) Quantification of acetylated H3 blots is presented as fold variations of the undifferentiated control (mean ± SD , n = 3). (C) The levels of acetylated H3 at the GATA4 promoter were determined by the ChIP analysis. Quantification is presented as fold variations of the undifferentiated control. (D) Occupancy of p300 at the GATA4 promoter was examined in parallel. (E) Quantification of the p300 Western blots is presented as fold variations of the undifferentiated controls (mean ± SD , n = 3).

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Western Blot, Negative Control, Control