Journal: Science Advances

Article Title: The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation

doi: 10.1126/sciadv.abj8360

Figure Lengend Snippet: ( A ) Schematic representation of ChIP-seq experiments after double treatment of dexamethasone and auxin of the HCT116 RAD21mAID cells. ( B ) Genome browser screenshot of representative GR-bound chromatin location in HCT116 RAD21mAID cells. Two biological replicates are combined for each sample. ( C ) Heatmap representing GR ChIP-seq intensity at GR chromatin–bound locations ( n = 371) identified in HCT116 RAD21mAID cells. Data combine two independent biological replicates. ( D ) Nascent RNA quantification before and after dexamethasone treatment before and after 6 hours of auxin treatment in RAD21mAID cells. Log 10 fold inductions (dexamethasone/EtOH) are normalized on GAPDH mRNA. Five biological replicates are shown for each column. Columns depict mean with error bars representing SD between experiments. P values derived from unpaired Mann-Whitney-Wilcoxon test. ( E to G ) YY1, H3K27ac ChIP-seq, and ATAC-seq in RAD21mAID cells, before and after auxin treatment. No substantial changes are found in YY1 chromatin binding and chromatin accessibility after acute depletion of cohesin subunit RAD21. Data combine two independent biological replicates each. All heatmaps and genomic data are normalized to a total of 10 million reads and further to local tag density. Homer and bedtools algorithms were used to identify unique and common sites between each experimental condition.

Article Snippet: The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160).

Techniques: ChIP-sequencing, Derivative Assay, MANN-WHITNEY, Binding Assay

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 1. LDB1 mediates chromatin contacts between cis-regulatory elements (A) Numbers of structural loops and CRE loops that are weakened, unchanged, or strengthened upon LDB1 depletion. Data shown for structural loops with LDB1 at 0 or 1 anchors. (B) Distribution of weakened CRE loop types. Fraction of loops with RAD21/CTCF co-occupied peaks in both anchors (below). (C) Fraction of enhancers and promoters in G1E-ER4 cells occupied by LDB1, YY1, and CTCF. (D) Schematic representing the motif analysis strategy for heterotypic loops and the top 10 most enriched motifs. (E) Change in loop strength upon LDB1 depletion for loops categorized based on LDB1 and CTCF occupancy. p values calculated using a two-sided Mann- Whitney U test. (F) LDB1-dependent homotypic loop (red arrow) and LDB1-dependent heterotypic loop (green arrow).

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: MANN-WHITNEY

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 2. LDB1-dependent CRE loops are associated with transcription activation (A) Gene expression changes measured by TT-seq upon LDB1 depletion (n = 3). (B) Gene expression changes (TT-seq) for genes categorized by the number of loop anchors overlapping their TSS. p values calculated using a two-sided Mann- Whitney U test. (C) Baseline gene expression measured by TT-seq. Genes categorized by the number of LDB1-dependent or -independent CRE loops they interact with. (D) Cumulative frequency distributions for gene distance to nearest LDB1 ChIP-seq peak. (E) Numbers of inter-TAD vs. intra-TAD LDB1-dependent CRE loops. (F) Loop lengths for LDB1-dependent inter-TAD and intra-TAD CRE loops. p values calculated using a two-sided Mann-Whitney U test. (G) Loop strengths for LDB1-dependent inter-TAD and intra-TAD CRE loops. Loop strength are calculated using 5k resolution, and p values are calculated using a two-sided Mann-Whitney U test.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: Activation Assay, Gene Expression, MANN-WHITNEY, ChIP-sequencing

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 3. LDB1 forms fine-scale looped networks at LDB1-dependent genes (A) Numbers of LDB1-dependent loops detected by Micro-C or RCMC. (B) Proportions of LDB1 or CTCF ChIP-seq peaks overlapping weakened loop anchors. (C) Examples of LDB1-dependent looped networks. (D) 10k resolution Tri-C contact maps for MYC proximal and distal regions. Contacts represent multi-way interactions involving the MYC promoter.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: ChIP-sequencing

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 4. At most locations, LDB1 occupancy is independent of YY1, CTCF, and cohesin and vice versa (A) LDB1 ChIP-seq peaks that are occupied by cohesin (RAD21), YY1, or CTCF. (B) LDB1-occupied enhancer elements that are occupied by cohesin (RAD21), YY1, or CTCF. (C) ChIP-seq profiles in LDB1-AID cells for RAD21, CTCF, and YY1 before/after LDB1 depletion. (D) RAD21 ChIP-seq signal at RAD21 ChIP-seq peaks overlapping CTCF peaks or LDB1 peaks. p values calculated using a two-sided Mann-Whitney U test. (E) ChIP-seq profiles in SMC3-AID, CTCF-AID, and YY1-AID cells before/after 4 h auxin treatment.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: ChIP-sequencing, MANN-WHITNEY

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 5. LDB1 can function in the absence of cohesin (A) Change in loop strength for LDB1-dependent CRE loops in response to LDB1 depletion (darker colors) or SMC3 depletion (lighter colors). p values calculated using a Wilcoxon signed-rank test. 3 LDB1-dependent loops were in sparse regions in SMC3-AID datasets and removed from analysis. (B) H3K27ac ChIP-seq signal at enhancers within LDB1-only loop anchors or dual-sensitive loop anchors. p values calculated using a two-sided Mann-Whitney U test. (C) Relative RNA levels for b-globin measured by RT-qPCR in SMC3-AID cells ± ZF-SA and ± auxin (4 h). p values calculated using one-way ANOVA. Data are represented as mean ± SD. (D) Lengths of LDB1 only and LDB1/cohesin dual sensitive loops.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: ChIP-sequencing, MANN-WHITNEY, Quantitative RT-PCR

Journal: Molecular cell

Article Title: LDB1 establishes multi-enhancer networks to regulate gene expression.

doi: 10.1016/j.molcel.2024.11.037

Figure Lengend Snippet: Figure 6. LDB1 chromatin occupancy correlates with loop establishment during G1-phase entry (A) ChIP-seq profiles for LDB1 at each cell-cycle stage at all LDB1 peaks identified in asynchronous cells. (B) APA plots from 10 kb resolution Hi-C data at each cell-cycle stage for each category of LDB1-dependent CRE loops. Average ChIP-seq profiles are shown for each loop type for LDB1 peaks within loop anchors. (C) Loop strength (top) and observed contacts between loop anchors (bottom) for each category of LDB1-dependent CRE loops and for structural loops at each cell-cycle stage. Median loop strength and observed contacts normalized to prometaphase are shown for each loop category. (D) Examples of an LDB1/cohesin dually sensitive loop and LDB1-only loop. The green arrow indicates the LDB1-dependent loop, and the blue arrow indicates an encompassing structural loop.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using the following antibodies: Pol2 (Cell Signaling, D8L4Y, 10uL/IP), LDB1 (Santa Cruz, sc-365074, 10ug/IP), CTCF (Millipore, 07-729, 10ug/IP), RAD21 (Abcam, ab992, 10ug/IP), YY1 (Active motif, 61779, 10ug/IP).

Techniques: ChIP-sequencing, Hi-C

Journal: Theranostics

Article Title: The kynurenine derivative 3-HAA sensitizes hepatocellular carcinoma to sorafenib by upregulating phosphatases

doi: 10.7150/thno.59841

Figure Lengend Snippet: 3-HAA inhibits AKT activity by upregulation of PPP1R15A. A. The genes involved in apoptosis pathway and upregulated by the combination of sorafenib and 3-HAA. The concentration of sorafenib and 3-HAA were 5 μM and 50 μM, respectively. B. The phosphatase PPP1α inhibitor okadaic acid but PPP2A inhibitor Calyculin A restored the AKT phosphorylation in 3-HAA-treated SMMC7721 cells. The dose of both calyculin A and okadaic acid were 2 nM. Cells were treated for 4 h. C. The PPP1R15A knockdown restores AKT phosphorylation suppressed by 3-HAA. The treating time and 3-HAA dose were the same as above. D. The effects of PPP1R15A/PPP1α on Akt phosphatase. The phosphatase assay was described in methods. E. The ChIP-sequencing analysis of YY1 on PPP1R15A gene. F. PPP1α inhibitor Okadaic acid (OA) partially restored the cell survival. The dose of Okadaic acid and the 3-HAA was 2 nM and 100 μM, respectively. Cells were treated for 24 h. G. The combination of sorafenib and 3-HAA dramatically decreased the tumor growth and tumor weights while PPP1R15A knockdown restored the xenograft growth and xenograft weights. Five mice were recruited in each group. H. The combination with 3-HAA treatment upregulated PPP1R15A expression and inhibited Akt activation in SMMC7721 xenografts. As xenografts reached at approximately 100 mm 3 , the sorafenib and/or 3-HAA were administered by intraperitoneal injection for 14 days at the dose of 100 mg/Kg.day and 30 mg/Kg.day, respectively.

Article Snippet: PARP1 (#13371-1-AP, dilutions 1:1000), PPP1R15A (#10449-1-AP, dilutions 1:500), (ProteinTech, Rosemont, IL, USA); PPP1α (#271762, dilation 1:1000) and β-actin (#47778, dilutions 1:2000), (Santa Cruz Biotechnology, Santa Cruz, CA, USA); YY1 (#61779, dilutions 1:500, Active Motif, CA, USA).

Techniques: Activity Assay, Concentration Assay, Phospho-proteomics, Knockdown, Phosphatase Assay, ChIP-sequencing, Expressing, Activation Assay, Injection

Journal: Theranostics

Article Title: The kynurenine derivative 3-HAA sensitizes hepatocellular carcinoma to sorafenib by upregulating phosphatases

doi: 10.7150/thno.59841

Figure Lengend Snippet: AKT inhibition is critical for 3-HAA sensitization of HCC to sorafenib. A. The Akt activator SC79 restored HCC cell growth inhibited by the combination of 3-HAA and sorafenib while the ERK activator BCI partially restored HCC cell growth. Cell viability was examined by the CCK8 method. The final concentration of sorafenib and 3-HAA was 5 μM and 50 μM, respectively. The final concentration of SC79 and BCI was 15 μM and 10 μM, respectively. B. The SC79 recovered tumor growth of SMMC7721 xenografts suppressed by the combination treatment. The tumor volumes are presented as mean ± SD. The dose of 3-HAA and sorafenib was 100 mg/Kg.day and 10 mg/Kg.day, respectively. The dose of SC79 was 40 mg/Kg.day. Five mice were recruited in each group. C. The apoptosis detection in xenografts by TUNEL assay. D. The working model for 3-HAA sensitizing sorafenib-resistant HCC cells. 3-HAA-upregulated PPP1R15A/PPP1α dephosphorylate/inactivates Akt which was compensatorily reactivated in sorafenib-resistant HCC cells, and consequently sensitized HCC cells to sorafenib.

Article Snippet: PARP1 (#13371-1-AP, dilutions 1:1000), PPP1R15A (#10449-1-AP, dilutions 1:500), (ProteinTech, Rosemont, IL, USA); PPP1α (#271762, dilation 1:1000) and β-actin (#47778, dilutions 1:2000), (Santa Cruz Biotechnology, Santa Cruz, CA, USA); YY1 (#61779, dilutions 1:500, Active Motif, CA, USA).

Techniques: Inhibition, Concentration Assay, TUNEL Assay

Journal: Cancer discovery

Article Title: Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

doi: 10.1158/2159-8290.CD-20-1848

Figure Lengend Snippet: Figure 4. A pharmacogenomic analysis identifies YY1-associated therapeutic response in brain cancers. A, Hierarchical clustering of drug AUC values from brain cancer cell lines. YY1 expression levels were shown on the top of the heatmap. Only drugs with Pearson correlation p-value < 0.01 were included. A low AUC value (blue) indicates sensitivity to drug treatment. B, Scatter plots showing correlation between YY1 expression levels and drug AUC values in brain cancer cell lines. Pearson correlation coefficients and p values were shown. C, Dose-response curves of transcriptional CDK inhibitors, alvocidib and dinaciclib, in YY1- dependent GSCs (GSC23, GSC1517 and GSC3264), YY1-independent GSCs (GSC1953 and MNK1) and non-malignant cells (astrocyte and HMEC). HMEC, Human Mammary Epithelial Cells. Data are presented as mean ± SD. D, Dose-response curves of cell cycle- related CDK4/6 inhibitor in 4 GSCs. Data are presented as mean ± SD. E-G, Dose-response curves of selective CDK9 inhibitors in GSCs. Data are presented as mean ± SD. H, Kaplan- Meier curves showing survival of immunocompromised mice bearing intracranial tumors

Article Snippet: The antibodies used were YY1 (Active Motif Cat# 61779, RRID:AB_2793763), CDK9 (Santa Cruz Biotechnology Cat# sc-484, RRID:AB_2275986; Santa Cruz Biotechnology Cat# sc-13130, RRID:AB_627245), phospho-RNA Polymerase II Ser2 (BETHYL Cat# A300–654A, RRID:AB_519341), β-Actin (Sigma Cat# A2228, RRID:AB_476697), BRD4 (Proteintech Cat# 67374–1- Ig, RRID:AB_2882622), AFF4 (Proteintech Cat# 14662–1-AP, RRID:AB_2242609), TRIM28 (Proteintech Cat# 15202–1-AP, RRID:AB_2209890).

Techniques: Clinical Proteomics, Expressing

Journal: Cancer discovery

Article Title: Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

doi: 10.1158/2159-8290.CD-20-1848

Figure Lengend Snippet: Figure 5. YY1 is involved in transcription elongation through interacting with CDK9 and other elongation factors. A, Enrichment map visualization of downregulated genes in GSEA after alvocidib treatment in GSC1517 cells. B, Bar plots showing relative expression levels of RNA processing and m6A genes after alvocidib treatment in GSC1517 determined by qRT-PCR. C, Bar plots showing relative m6A levels after alvocidib treatment in GSC1517 and GSC23. D-F, Bar plots showing relative expression levels of METTL3 and YTHDF2 after treatment of selective CDK9 inhibitors in GSC1517 determined by qRT-PCR. G, Immunoblot showing Ser2 phosphorylation (Ser2p) of RNA Pol II CTD (C-terminal domain) in GSC after alvocidib treatment. Data are representative results of three independent experiments. H, Immunoblot showing YY1 and Ser2 phosphorylation (Ser2p) of RNA Pol II CTD in GSCs after YY1 knockdown. I, Empirical cumulative density function (ECDF) plots of Pol II pausing index after YY1 knockdown in GSCs. J and K, Visualization of Pol II occupancy at representative genomic loci, METTL3 (J) and HNRNPU (K). L, Co-immunoprecipitation

Article Snippet: The antibodies used were YY1 (Active Motif Cat# 61779, RRID:AB_2793763), CDK9 (Santa Cruz Biotechnology Cat# sc-484, RRID:AB_2275986; Santa Cruz Biotechnology Cat# sc-13130, RRID:AB_627245), phospho-RNA Polymerase II Ser2 (BETHYL Cat# A300–654A, RRID:AB_519341), β-Actin (Sigma Cat# A2228, RRID:AB_476697), BRD4 (Proteintech Cat# 67374–1- Ig, RRID:AB_2882622), AFF4 (Proteintech Cat# 14662–1-AP, RRID:AB_2242609), TRIM28 (Proteintech Cat# 15202–1-AP, RRID:AB_2209890).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Knockdown, Immunoprecipitation

Journal: Cancer discovery

Article Title: Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

doi: 10.1158/2159-8290.CD-20-1848

Figure Lengend Snippet: Figure 6. Targeting transcription elongation complexes elicits interferon response in glioblastoma stem cells. A, GSEA plots of representative gene sets involved in immune response after alvocidib treatment. The normalized enrichment score (NES) and false discovery rate (FDR) were indicated. B, Heatmap showing immune response related differentially expressed genes after alvocidib treatment in GSC1517 cells. C, Bar plots showing expression levels of IFNα and IFNβ in two GSC models (GSC1517 and GSC23) with alvocidib treatment determined by qRT-PCR. D and E, Bar plots showing expression levels of interferon-stimulated genes with alvocidib treatment in GSC1517 determined by qRT-PCR. F-H, Bar plots showing expression levels of IFNα and IFNβ in two GSC models (GSC1517 and GSC23) with selective CDK9 inhibitor treatment determined by qRT-PCR. I and J, Bar plots showing expression levels of IFNα and IFNβ in two GSCs (GSC1517 and GSC23) with BEC inhibitor JQ1 (I) and SEC inhibitor KL-2 (J) treatment determined by qRT-PCR. Data are

Article Snippet: The antibodies used were YY1 (Active Motif Cat# 61779, RRID:AB_2793763), CDK9 (Santa Cruz Biotechnology Cat# sc-484, RRID:AB_2275986; Santa Cruz Biotechnology Cat# sc-13130, RRID:AB_627245), phospho-RNA Polymerase II Ser2 (BETHYL Cat# A300–654A, RRID:AB_519341), β-Actin (Sigma Cat# A2228, RRID:AB_476697), BRD4 (Proteintech Cat# 67374–1- Ig, RRID:AB_2882622), AFF4 (Proteintech Cat# 14662–1-AP, RRID:AB_2242609), TRIM28 (Proteintech Cat# 15202–1-AP, RRID:AB_2209890).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer discovery

Article Title: Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

doi: 10.1158/2159-8290.CD-20-1848

Figure Lengend Snippet: Figure 7. Pharmacological Targeting of the YY1-CDK9 complex reprograms microenvironment and enhances anti-PD-1 response in gliomas. A, The workflow of the cytometry by time of flight (CyTOF) experiment to analyze the microenvironment in GBM models. B, The t-SNE plots of immune cell clusters from GL261 tumors treated with vehicle, alvocidib, anti-PD1 and combination, colored by cell clusters, from CyTOF analysis. C, Proportion of cells from GL261 tumors for the indicated cell types. D and E, Kaplan-Meier curves showing survival of immunocompetent mice bearing intracranial tumors from syngeneic CT2A (D) and GL261 (E) cells with alvocidib and anti-PD-1 treatment. *p < 0.05, **p < 0.01 by log-rank test; ns, not significant. F, In vivo bioluminescent imaging of immunocompetent mice bearing intracranial syngeneic tumors from mouse glioma CT2A cells with alvocidib and anti-PD-1 treatment. G, Kaplan-Meier curves showing survival of immunocompetent mice from Fig. 7D re-challenged with GL261 cells. P value was determined by log-rank test. H, Box-and-whisker plot showing expression pattern of YY1 in normal brains, low grade gliomas and glioblastomas from TCGA and

Article Snippet: The antibodies used were YY1 (Active Motif Cat# 61779, RRID:AB_2793763), CDK9 (Santa Cruz Biotechnology Cat# sc-484, RRID:AB_2275986; Santa Cruz Biotechnology Cat# sc-13130, RRID:AB_627245), phospho-RNA Polymerase II Ser2 (BETHYL Cat# A300–654A, RRID:AB_519341), β-Actin (Sigma Cat# A2228, RRID:AB_476697), BRD4 (Proteintech Cat# 67374–1- Ig, RRID:AB_2882622), AFF4 (Proteintech Cat# 14662–1-AP, RRID:AB_2242609), TRIM28 (Proteintech Cat# 15202–1-AP, RRID:AB_2209890).

Techniques: Cytometry, In Vivo, Imaging, Whisker Assay, Expressing

Journal: Science Advances

Article Title: The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation

doi: 10.1126/sciadv.abj8360

Figure Lengend Snippet: ( A ) GR-cohesin–interacting partners identified by GR ChIP-SICAP. GR interacts significantly with NIPBL and SMC1a, marked in red. ( B ) GR-immunoprecipitation (and FLAG-IP) and blotting against SMC1, SMC3, NIPBL, and GR after 1 hour of EtOH or dexamethasone treatment in mouse breast adenocarcinoma cells. ( C ) Endogenous interaction, measured by proximity ligation assay, between GR and NIPBL (left) and SMC3 (right). ( D ) GR, NIPBL, and SMC1a ChIP-seq after 1 hour of EtOH or dexamethasone treatment. ( E ) Heatmaps representing GR, NIPBL, and SMC1a ChIP-seq at the 3000 strongest GR locations previously identified in 3134 cells before and after 1 hour of 100 nM dexamethasone. Data combine two biological replicates normalized to 10 million reads. ( F ) Heatmap of GR ChIP-seq intensity at the 3000 strongest GR locations [same as (E)] in siCTRL, siSMC1a, and siNIPBL cells. Genomic data are normalized [fragments per kilobase million (fpkm)] to a total of 10 million reads and further to local tag density ( P < 0.000001 from Wilcoxon test). ( G ) Venn diagram of GR ChIP-seq peaks identified in two independent replicates of siCTRL, siSMC1a, and siNIPBL cells. ( H ) Genome browser screenshot of the Tsc22d3 gene locus for the ChIP-seq of GR, NIPBL, SMC1A, RAD21, and CTCF. The TAD boundary at the 3′ untranslated region of TSC22D3 gene was used to evaluate cohesin binding and loop extrusion mechanism mediated by GR. ( I ) Red highlighted regions correspond to PCR products designed to evaluate the loop extrusion process. On the right side, ChIP qRT-PCR for SMC1a after EtOH treatment and 20′ and 60′ of dexamethasone (data points show three biological replicates).

Article Snippet: The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160).

Techniques: Immunoprecipitation, Proximity Ligation Assay, ChIP-sequencing, Binding Assay, Quantitative RT-PCR

Journal: Science Advances

Article Title: The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation

doi: 10.1126/sciadv.abj8360

Figure Lengend Snippet: ( A ) Chromosome contact maps for three successive zoom-ins across mouse chromosome 1 using Micro-C data. Red arrows depict anchors by moustache loop calling, and red boxes are architectural stripes detected by Stripenn. ( B ) Middle: Total numbers of Micro-C loops in siCTRL EtOH and siCTRL Dex. A total of 2650 loops were identified as dex-induced while 6350 were found as dex-repressed loops. siNIPBL cells showed a reduction of the dex-induced loops: measured by the ratio of the dex-induced loops over the sum of the differential loops and found by comparing siCTRL EtOH versus siCTRL Dex and siNIPBL EtOH versus siNIPBL Dex. There was no change in the dex-repressed loops as measured by the ratio of the dex-repressed loops. ( C ) Micro-C robustly captures enhancer-promoter contacts driven by the GR. GR, SMC1A, NIPBL, and CTCF ChIP-seq are on top of the contact maps. All normalized datasets represent two biological replicates and plotted at 2-kb resolution. ( D ) Aggregate peak analysis plots of the dex-induced loops. The heatmaps below show the GR ChIP-seq intensity at the dex-induced interactions. ( E ) Contact frequencies, in siCTRL Dex, of the architectural stripe connecting the GR-bound superenhancer to the gene Nsmce2 . ( F ) GR and NIPBL promote the formation of dexamethasone-dependent architectural stripes. Boxplot depicting the significance [−log 10 ( P value)] of all the GR-bound architectural stripes ( n = 561), in siCTRL EtOH, siCTRL dex, siNIPBL EtOH, and siNIPBL dex Micro-C data. ( G ) Heatmap showing the intensity of the architectural stripes (two biological replicates were combined on the level of binned data).

Article Snippet: The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160).

Techniques: ChIP-sequencing

Journal: Science Advances

Article Title: The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation

doi: 10.1126/sciadv.abj8360

Figure Lengend Snippet: ( A ) Schematic illustration of HCR protocol. ( B ) Representative images of nascent RNA before and after Dex treatment. ARL4D images are taken after 1 hour of dexamethasone; TSC22D3 images are taken after 4 hours of Dex treatment. Scale bar, 5 μm. ( C ) Average number of active nascent RNA foci per well at 0, 1, and 4 hours of dex treatment for ARL4D and TSC22D3, after 72 hours of siRNA against SMC1a or RAD21. Left: TSC22D3; right: ARL4D. TSC22D3 P value is 0.012 for siRAD21 and 0.0005 for siSMC1a, both derived from two-way analysis of variance (ANOVA) test. ARL4D, P < 0.00001 derived from two-way ANOVA test both for siSMC1a and siRAD21. ( D ) Nascent RNA intensity of each of the foci in control cells and SMC1a and RAD21 knockdown cells. Left: TSC22D3; right: ARL4D. P < 0.00001 for TSC22D3 and P < 0.001 for ARL4D, derived from two-way ANOVA test for siSMC1a and siRAD21. No statistically significant difference was found between siSMC1a and siRAD21 cells. ( E ) Percentage of actively transcribing cells measured by HCR before and after dex treatment in control and SMC1 and RAD21 knockdown cells. Left: TSC22D3; right: ARL4D. At 0-hour treatment, almost 100% of cells are not actively transcribing. After 1 or 4 hours of dexamethasone treatment, approximately 70% of control cells transcribe nascent RNA of the target genes. In siSMC1a or siRAD21 cells, only 30% of cells actively transcribe nascent RNA for GR-regulated genes. siGR cells, the negative control, show no gene activation after dexamethasone treatment, confirming that nascent RNA foci enrichment is GR dependent.

Article Snippet: The following are the antibodies used in this study: NIPBL (Bethyl Laboratories, A301-779A; Thermo Fisher Scientific, MA1-72534), YY1 (Active Motif, 61779), H3K27ac (Active Motif, 39133), GR (Santa Cruz Biotechnology, sc-393232; Thermo Fisher Scientific, MA1-510), SMC1a (Bethyl Laboratories, A300-055A; Abcam, ab133643), SMC3 (Abcam, ab9263), RAD21 (Abcam, ab992), H2B (Abcam, ab61250), and tubulin (Abcam, ab6160).

Techniques: Derivative Assay, Control, Knockdown, Negative Control, Activation Assay