Journal: bioRxiv

Article Title: PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

doi: 10.1101/2022.12.27.521987

Figure Lengend Snippet: A. Western blot analyses showing SMC1-non-tagged and SMC1-EGFP tagged cells (left, n=2), and PAXIP1 expression in SMC1-EGFP tagged cells, with actin as control, in NT-SMC1-EGFP and PAXIP1 -KO-SMC1-EGFP (n =2). B. Pie-charts representing calculated fraction sizes of mobile, and short, intermediate and long fractions attributed to SMC1-EGFP, in NT and PAXIP1-KO cells, in both DMSO and GC-treated cells. Monte Carlo simulation calculations were performed to calculate each fraction. C. Average density plot of RAD21 (left), PAXIP1 (middle) and H3K4me1 (right) ChIP-seq signal in NT and PAXIP1 -KO cells in vehicle or GC-treated conditions, depicting a ±2.5kb window around the peak center. Data represent the average of two biological replicates. D. Genomic distribution (top) and distance to TSS (bottom) of GR-PAXIP1-RAD21 commonly bound sites. E. Motif analyses of GR-PAXIP1-RAD21 sites. Font size represents z-score. Motifs are colored according to different families of transcription factors. F. RAD21 genomic binding distribution in NT and PAXIP1 -KO under GC-treated conditions. G. Motif analyses of RAD21-binding sites across all the genome. Font size represents z-score. Motifs are colored according to different families of transcription factors. H. ChIP-seq tracks at FKBP5 locus depicting GR and H3K4me1 binding following GC treatment for 2 hours. Data is the merge of three (GR) and two (H3K4me1) independent biological replicates. A. 4C-seq experiments in NT and PAXIP1 -KO cells using the FKBP5 TSS as a viewpoint showing DMSO and GC-treated conditions. 4C data is shown as the mean of three independent biological experiments. ChIP tracks of RAD21 and GR in GC-treated cells are depicted. ChIP-tracks are a representative snapshot of one biological replicate.

Article Snippet: The antibodies that were used are the following: GR (12041, Cell Signaling Technology), RAD21 (05-908, Merck), PAXIP1 (ab70434, abcam), H3K4me1 (ab8895, abcam).

Techniques: Western Blot, Expressing, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

doi: 10.1101/2022.12.27.521987

Figure Lengend Snippet: A. Representative image of FRAP experiments in NT-SMC1-EGFP and PAXIP1 -KO-SMC1-EGFP cells with vehicle (top, blue) and GC-treated (bottom, red) cells. B. Quantification of FRAP experiments over time in NT-SMC1-EGFP and PAXIP1 -KO-SMC1-EGFP cells with vehicle (above) and GC-treated cells (below). Average ± SD for 30 cells quantified over 3 independent biological experiments. Intensity is pre-bleached normalized. C. Heatmap of RAD21, PAXIP1 and H3K4me1 ChIP-seq signal in NT, PAXIP1 -KO cells in vehicle (DMSO) or treated (GC) conditions. Data are centered at commonly bound RAD21 and GR-peaks in NT cells treated with GC, depicting a ±10kb window around the peak center (n=2). D. Snapshots of GR, RAD21 and PAXIP1 ChIP-seq tracks around the FKBP5 locus. E. 4C-seq experiments in NT and PAXIP1 -KO cells using the FKBP5 TSS as a viewpoint. Cells were treated with GCs for 2 hours. 4C data is shown as the mean of three independent biological experiments. ChIP tracks of RAD21 and GR in GC-treated cells are depicted. ChIP-tracks are a representative snapshot of one biological replicate.

Article Snippet: The antibodies that were used are the following: GR (12041, Cell Signaling Technology), RAD21 (05-908, Merck), PAXIP1 (ab70434, abcam), H3K4me1 (ab8895, abcam).

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

doi: 10.1101/2022.12.27.521987

Figure Lengend Snippet: A. MA plots depicting differential gene expression between DMSO- and GC-treatment in NT, PAXIP1 -KO and STAG2 -KO cells, NT vs PAXIP1 -KO in GC-treated cells, and NT vs STAG2 -KO in GC-treated cells. Red and blue dots indicate significantly differentially expressed genes with log 2 (fold change)>1 and adjusted p-value <0.05. Data represent the average of three independent biological replicates. B. Correlation of differentially expressed genes in STAG2 -KO cells with PAXIP1 -KO cells, both in GC-treated conditions. Genes differentially expressed in PAXIP1 -KO cells (padj < 0.05) were ranked based on log 2 (fold change). C. sevenC analyses of WT and PAXIP1 -KO based on RAD21 ChIP-seq and CTCF motifs. Number of predicted genomic interactions per chromosome in GC-treated conditions are depicted. One sample t-test was used. P-value < 0.0001 D. Snapshot of sevenC predicted chromosomal interactions in chr 11, both in NT and PAXIP1 -KO cells.

Article Snippet: The antibodies that were used are the following: GR (12041, Cell Signaling Technology), RAD21 (05-908, Merck), PAXIP1 (ab70434, abcam), H3K4me1 (ab8895, abcam).

Techniques: Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

doi: 10.1101/2022.12.27.521987

Figure Lengend Snippet: A. Significantly downregulated genes with a log 2 (fold change)>1, and p-adjusted <0.05, in PAXIP1 -KO and STAG2 -KO cells treated with GC when compared to NT GC-treated cells. Significant overlap was calculated with a Fisheŕs exact t-test. B. Snapshot of sevenC prediction around the P4HA3 and PPME1 loci, both in NT and PAXIP1 -KO cells following GC-treatment. C. Normalized P4HA3 (left) and PPME1 (right) expression in NT, PAXIP1 -KO and GR -KO cells (n = 3). p-value was calculated using U Mann-Whitney t-test. D. 4C-seq experiments in NT and PAXIP1 -KO cells using the P4HA3 TSS as a viewpoint showing NT vs PAXIP1 -KO in GC-treated conditions. 4C data is shown as the mean of three independent biological experiments. ChIP tracks of RAD21 and GR in GC-treated cells are depicted. ChIP-tracks are a representative snapshot of one biological replicate.

Article Snippet: The antibodies that were used are the following: GR (12041, Cell Signaling Technology), RAD21 (05-908, Merck), PAXIP1 (ab70434, abcam), H3K4me1 (ab8895, abcam).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

doi: 10.1101/2022.12.27.521987

Figure Lengend Snippet: A. Cell proliferation of NT and PAXIP1 -KO cells following DMSO or GC treatment over time (n = 3 up to 168 hrs, n = 2 from 168hrs to 216hrs, error bars represent 95% confidence interval) B. Overlap of significantly differentially expressed genes in PAXIP1 -KO and STAG2 -KO cells following GC stimulation (log 2 (FoldChange) > 0.75; padj < 0.05), with a cell cycle gene set (hsa04110). C. Boxplot showing normalized CDKN1B expression in NT, PAXIP1 -KO and GR -KO cells upon vehicle or GC treatment for 8 hours (n =3). p-value was calculated using U Mann-Whitney t-test. D. P27 protein expression levels in NT, PAXIP1 -KO and GR -KO cells upon vehicle or GC treatment for 24 hours. Actin was used as a loading control (n = 2). E. Cell proliferation of PAXIP1 -KO cells and PAXIP1 / CDKN1B -DKO following DMSO or GC treatment over time (n = 3 up to 168hrs, n = 2 from 168hrs to 216hrs, error bars represent 95% confidence interval). F. 4C-seq experiments in NT and PAXIP1 -KO cells using the CDKN1B TSS as a viewpoint showing NT vs PAXIP1 -KO in GC-treated conditions. Cells were treated for 2 hours with GCs. 4C data is shown as the mean of three independent biological experiments. ChIP tracks of RAD21 and GR in GC-treated cells are depicted. ChIP-tracks are a representative snapshot of one biological replicate.

Article Snippet: The antibodies that were used are the following: GR (12041, Cell Signaling Technology), RAD21 (05-908, Merck), PAXIP1 (ab70434, abcam), H3K4me1 (ab8895, abcam).

Techniques: Expressing, MANN-WHITNEY

Journal: Cell

Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

doi: 10.1016/j.cell.2017.12.021

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Rad21 (fission yeast) , BioAcademia , Cat# 63-139.

Techniques: Recombinant, Protease Inhibitor, Staining, Cloning, Western Blot, Plasmid Preparation, Software

Journal: EMBO Reports

Article Title: STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1

doi: 10.1038/s44319-024-00303-6

Figure Lengend Snippet: ( A ) Chromatin-bound levels of cohesin subunits and regulators assessed by Chromoflow flow cytometry. A representative experiment comparing A673 WT and STAG2 KO#1 is shown. Similar results were obtained comparing WT or Parental cells and STAG2 KO#1 or KO#2 in two independent experiments. ( B ) Immunoprecipitation of cohesin with anti-SMC1 from the indicated cell extracts followed by immunoblotting of cohesin and regulators. Colored dots below the panels identify each sample. ( C ) Quantification of the amount of the indicated proteins co-immunoprecipitated with anti-SMC1, relative to the amount of RAD21, in these four samples. Gray dots often overlap with black dots. .

Article Snippet: Mouse monoclonal anti-RAD21 clone 53A303; FC: 2 µg/ml , Merck , cat#05-908.

Techniques: Flow Cytometry, Immunoprecipitation, Western Blot

Journal: EMBO Reports

Article Title: STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1

doi: 10.1038/s44319-024-00303-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse monoclonal anti-RAD21 clone 53A303; FC: 2 µg/ml , Merck , cat#05-908.

Techniques: Recombinant, Sequencing, Reverse Transcription, SYBR Green Assay, Software, Isolation