gtf3c1 novus biologicals  (Novus Biologicals)

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet:

Article Snippet: rabbit anti-GTF3C1/TFIIIC220 Antibody, Affinity Purified WB , Bethyl , RRID: AB_938042.

Techniques: Affinity Purification, Virus, Recombinant, Protease Inhibitor, Sequencing, Multiplex Assay, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Electrophoresis, Gene Expression, Expressing, Plasmid Preparation, Software

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet:

Article Snippet: rabbit anti-GTF3C1/TFIIIC220 Antibody, Affinity Purified WB , Bethyl , RRID: AB_938042.

Techniques: Affinity Purification, Virus, Recombinant, Protease Inhibitor, Sequencing, Multiplex Assay, Bicinchoninic Acid Protein Assay, cDNA Synthesis, SYBR Green Assay, Electrophoresis, Gene Expression, Expressing, Plasmid Preparation, Software

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet: ADNP Guides TFIIIC Selective Recruitment to AEs and TFIIIC-Associated Promoters (A) Area plot ranking unique spectral counts (average of three replicates) of proteins identified in a TFIIIC-RIME experiment using GTF3C2 as bait in T47D cells grown in the presence (+S) or absence of serum (–S). Data from control immunoprecipitations using normal IgG in the presence of serum are also included (blue). Prey proteins belonging to the ChAHP complex are shown in purple. (B) Direct interaction of recombinant ADNP and TFIIIC. For TFIIIC expression, insect cells were infected with a single baculovirus expressing GTF3C1-3XFLAG and the rest of the TFIIIC subunits (GTF3C2-6) untagged. Left panel: Coomassie staining of anti-FLAG immuno-purifications of insect cell lysates overexpressing FLAG-TFIIIC (lane 1), HA-ADNP (lane 2), or both simultaneously (lane 3). Note that GTF3C1 immunopurification via the FLAG-tag pulled down the whole TFIIIC complex (lane 1). ADNP co-purifies with TFIIIC (lane 3). GTF3C5 fractionates on top of the heavy IgG chain (IgG H ). Right panel: Coomassie staining of anti-HA immunopurification of lysates expressing FLAG-TFIIIC alone (lane 4), HA-ADNP alone (lane 5), or both together (lane 6). The presence of the TFIIIC subunits is detected in the ADNP immuno-complexes. The position of the IgG heavy and light chains (IgG H and IgG L , respectively) is indicated. The identity of the bands was confirmed by mass spectrometry and western blotting with HA and GTF3C2 antibodies ( Figure S2 A). (C) Heatmap of ADNP-eGFP binding in K562 cells (GEO: GSE105573 ) spanning ±1 kb across all TFIIIC-bound AEs in T47D, ranked from high to low ADNP-eGFP enrichment. Color bar scale with increasing shades of color stands for higher enrichment (–log10 of the Poisson p value). (D) Venn diagram with the number of TFIIIC peaks upon ADNP depletion (siADNP, yellow) compared to control cells (siCTRL, blue) in serum-starved T47D cells. (E) Sitepro profile of TFIIIC occupancy in control (siCTRL) and ADNP-depleted cells (siADNP) in serum-starved T47D cells. The graphs are plotted over the summit of TFIIIC peaks in the siCTRL cells (p value, two-tailed unpaired t test). ADNP depletion levels are shown in E and S2F. (F) Genome browser view of representative TFIIIC-associated genes FEM1A and HELLS showing TFIIIC occupancy in ADNP-depleted (siADNP) and T47D control cells (siCTRL). ADNP binding from GEO: GSE105573 is represented by the blue track (eGFP-ADNP). The gene structure and the direction of transcription (arrow) are shown at the bottom.

Article Snippet: Commercial antibodies were: CTCF, Millipore (07-729); Pol II (NTD, D8L4Y), Cell Signaling (14958); TFIIIC63 ( GTF3C5 ), Bethyl (A301-242A); TFIIIC220 ( GTF3C1 ), Novus Biologicals (NB100-60657), Bethyl (A301-293A) for western blotting and Bethyl (A301-291A) for ChIP; TFIIIC90 ( GTF3C4 ), Abcam (ab74229); α-Tubulin ( TUBA4A ), Sigma (T9026); ADNP, Abcam (ab54402); histone H1.2, Abcam (ab4086); H3K27ac, Millipore (17-683); p300 ( EP300 ), Santa Cruz (sc584-Lot: J0915 and sc585-Lot: I2815).

Techniques: Control, Recombinant, Expressing, Infection, Staining, Immu-Puri, FLAG-tag, Mass Spectrometry, Western Blot, Binding Assay, Two Tailed Test

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet: TFIIIC-Associated HAT Activity Acetylates H3K18 at Nucleosomes over AEs in Response to Serum Depletion (A) ATAC-seq signal enrichment in T47D grown in the presence (+S) or in the absence of serum (–S) across TFIIIC-bound AEs detected in the absence of serum (–S) (–log10 of the Poisson p value). p value, two-tailed t test comparing each condition to its random control. (B) Average profile of H3K18ac enrichment in T47D grown in the presence (+S,) or absence of serum (–S) across all TFIIIC-bound AEs (–log10 of the Poisson p value). p value, two-tailed paired t test. (C) Sitepro average plot of nucleosome signals derived from ATAC-seq by selecting sequenced fragments of 180–250 bp ( Buenrostro et al., 2013 ) spanning a 1 kb region over all the AEs bound by TFIIIC in T47D grown in the presence (+S) or absence (–S) of serum. Note the presence of a peak in the center of the region representing the AE (gray rectangle). SS causes a decrease in the nucleosome signal for both the AE-nucleosome and the surrounding ones (two-tailed unpaired t test). (D) Genome browser view of representative cell-cycle-related TFIIIC-associated promoters with normalized counts of ATAC-derived nucleosome signal for T47D grown in the presence (+S) or absence (–S) of serum. The position of the AE bound by TFIIIC positive for H3K18ac is indicated by a yellow rectangle. AE nucleosomes reduced upon SS are highlighted by a gray rectangle. (E) Venn diagram showing the total number of AEs bound by TFIIIC and those acetylated in H3K18 in serum-starved T47D. (F) Venn diagram showing the total number of TFIIIC-associated Pol II promoters and those enriched in H3K18ac in T47D grown in the absence of serum. (G) Plot for p300 occupancy across all TFIIIC-bound AEs spanning a 6 kb region in T47D grown in the presence (+S) or absence (–S) of serum. (H) H3K18ac immunostaining (red) in serum-starved T47D cells knocked down for GTF3C1 (siGTF3C1) or control (siCTRL). DAPI was used to stain nuclei (blue). Two different fields are shown. Scale bar, 100 μm. GTF3C1 depletion levels are shown in Figure S3 H and quantification in Figure S3 I.

Article Snippet: Commercial antibodies were: CTCF, Millipore (07-729); Pol II (NTD, D8L4Y), Cell Signaling (14958); TFIIIC63 ( GTF3C5 ), Bethyl (A301-242A); TFIIIC220 ( GTF3C1 ), Novus Biologicals (NB100-60657), Bethyl (A301-293A) for western blotting and Bethyl (A301-291A) for ChIP; TFIIIC90 ( GTF3C4 ), Abcam (ab74229); α-Tubulin ( TUBA4A ), Sigma (T9026); ADNP, Abcam (ab54402); histone H1.2, Abcam (ab4086); H3K27ac, Millipore (17-683); p300 ( EP300 ), Santa Cruz (sc584-Lot: J0915 and sc585-Lot: I2815).

Techniques: Activity Assay, Serum Depletion, Two Tailed Test, Control, Derivative Assay, Immunostaining, Staining

Journal: Molecular Cell

Article Title: TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation

doi: 10.1016/j.molcel.2019.10.020

Figure Lengend Snippet:

Article Snippet: Commercial antibodies were: CTCF, Millipore (07-729); Pol II (NTD, D8L4Y), Cell Signaling (14958); TFIIIC63 ( GTF3C5 ), Bethyl (A301-242A); TFIIIC220 ( GTF3C1 ), Novus Biologicals (NB100-60657), Bethyl (A301-293A) for western blotting and Bethyl (A301-291A) for ChIP; TFIIIC90 ( GTF3C4 ), Abcam (ab74229); α-Tubulin ( TUBA4A ), Sigma (T9026); ADNP, Abcam (ab54402); histone H1.2, Abcam (ab4086); H3K27ac, Millipore (17-683); p300 ( EP300 ), Santa Cruz (sc584-Lot: J0915 and sc585-Lot: I2815).

Techniques: Affinity Purification, Virus, Recombinant, Protease Inhibitor, Sequencing, Multiplex Assay, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Electrophoresis, Expressing, Plasmid Preparation, Software