Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Inhibition of BRD9 enhances the induction of PPARα-downstream gene CPT1A by WY-14643. A: Schematic image of mammalian cBAF, PBAF, and ncBAF . B: Glycerol gradient density sedimentation of the nuclear extract of HepG2 cells. Subunits of each SWI/SNF complex and PPARα protein were detected by Western blot analysis (C–G) HepG2, PHH, PMH, or HepaSH cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. Human (C and E) and mouse (F) CPT1A mRNA levels were determined using real-time RT-PCR. (D) CPT1A, PPARα, and GAPDH protein levels in HepG2 cells were determined by Western blotting. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. In panel (C), data were analyzed using one-way ANOVA followed by the Games-Howell test. In panel (F), data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with NT. † P < 0.05, †† P < 0.01 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Inhibition, Sedimentation, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of BRD7 and BRD9 knockdown on CPT1A expression in WY-14643-treated HepG2 cells. A–C: HepG2 cells were transfected with siRNA against BRD7 (siBRD7) or BRD9 (siBRD9). After 24 h, the cells were treated with 30 μM WY-14643 for 48 h. A: BRD7, BRD9, PPARα, and GAPDH protein levels were determined using Western blot analysis. B and C: CPT1A mRNA and protein levels were determined using real-time RT-PCR and Western blotting analysis, respectively. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001, compared with NT, † P < 0.05 and ††† P < 0.001, compared with siControl. NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: BRD9 directly interacts with PPARα. A: Co-immunoprecipitation assay to examine the interaction between PPARα and BRD9 was performed. HEK293T cells were transfected with FLAG-PPARα plasmid together with the BRD7-His or BRD9-His plasmids using Lipofectamine 3000. Cell lysates were subjected to co-immunoprecipitation with His-tag antibody. B: Direct interaction between PPARα and BRD9. HEK293T cells were individually transfected with FLAG-PPARα, BRD7-His, or BRD9-His plasmids. Affinity purification was conducted using cell lysates, and the purified FLAG-PPAR protein was incubated with purified BRD7/9-His proteins. C: Western blotting to analyze lysine acetylation of PPARα. Lysate of FLAG-PPARα plasmid-transfected HEK293T cells was subjected to Western blotting using anti-acetylated lysine and anti-FLAG antibodies. A smaller amount of protein (10 μg) was loaded compared to A (30 μg). Western blot experiments were conducted with three independent replicates.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Affinity Purification, Purification, Incubation, Western Blot

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of WY-14643 and/or BI-9564 treatment on the binding of PPARα and BRD9 and on the chromatin accessibility around the CPT1A intronic peroxisomal proliferator response element (PPRE). A: Scheme of the CPT1A gene. B: Binding of PPARα to CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. A ChIP assay using an anti-PPARα antibody was performed, and the purified DNA was analyzed by real-time PCR targeting CPT1A intronic PPRE. C: The interaction between PPARα and BRD9. HEK293T cells were transfected with FLAG-PPARα and BRD9-His plasmids using Lipofectamine 3000, and treated with 30 μM WY-14643 and/or 40 μM BI-9564 for 48 h. The cell lysates were co-immunoprecipitated with anti-His-tag antibody. FLAG-PPARα and BRD9-His were detected by Western blotting. D: Chromatin accessibility around CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h; a FAIRE assay was then performed on the cells. Purified DNA was analyzed by real-time PCR targeting the CPT1A intronic PPRE. Each column represents the mean ± SD (n = 3). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗∗ P < 0.001, compared with NT; † P < 0.05 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Binding Assay, Purification, Real-time Polymerase Chain Reaction, Transfection, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) qRT-PCR analysis of Brg1, Cdx2, and Oct4 transcripts in Brg1 KD 8-cell embryos, morulae, and blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control embryos at each stage; black line = 1. Asterisk denotes significant difference between Brg1 KD and control blastocysts (p<0.05). (B) ICC analysis of Oct4 and Cdx2 expression in Brg1 KD 8-cell embryos, morulae, and blastocysts. Nuclei were counter stained with DAPI (blue). (C) Brg1 represses Oct4 expression in a dose dependent manner. One-cell embryos were injected with 0 µM (control), 0.1 µM, 1 µM, or 100 µM Brg1 siRNA and cultured to the blastocyst stage. Real-time qPCR was used to analyze the levels of Brg1 and Oct4 transcripts. Data were normalized to Ubtf and are relative to control blastocysts; dashed line = 1.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Quantitative RT-PCR, Control, Expressing, Staining, Injection, Cell Culture

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) ICC analysis of Oct4 and Cdx2 in Brg1 KD and control blastocysts. In control blastocysts (a–d) Oct4 expression (green) is restricted to the ICM and is largely absent in the Cdx2-positive (red) trophectoderm. In contrast, in Brg1 KD blastocysts (e–h) Oct4 is widely expressed in both the ICM and cdx2-positive (yellow) trophectoderm. Arrowheads denote co-expression of Oct4 and Cdx2. Nuclei were counter stained with DAPI (blue). (B) Quantification of the average number of cells in Brg1 KD and control blastocysts expressing Oct4, Cdx2, and Oct4 & Cdx2 (double expression). Asterisks denote statistical significance (p<0.05) between Brg1 KD and control blastocysts. A total of 25 Brg1 KD blastocysts and 15 control blastocysts were analyzed.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Control, Expressing, Staining

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) Combined depletion of Brg1 and Cdx2 augments Oct4 expression in blastocysts. qRT-PCR analysis of Oct4 transcripts in Brg1 KD blastocysts, Cdx2 KD blastocysts, and Brg1 & Cdx2 double KD blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control blastocysts; dashed line = 1. Different letters denote statistical significance in Oct4 transcripts (p<0.05). These experiments were replicated using a total of 5 biological replicates. (B) Co-immunoprecipitation and western blot analysis of Brg1 and Cdx2 in TS cells. Brg1 was immunoprecipitated using a rabbit anti-serum. Recovery of Cdx2 was measured by western blot analysis. Cdx2 is enriched in the Brg1 IP samples and not in the control IgG samples. This assay was repeated a total of 4 times using different batches of TS cells. (C) Confocal immunofluorescence analysis of Brg1 and Cdx2 in blastocysts. Co-localization of endogenous Brg1 and Cdx2 in trophectoderm nuclei was determined using specific antibodies for Brg1 and Cdx2. Nuclei were counterstained with DAPI. White box represents magnified region in bottom panel. Arrow denotes blastocyst ICM. (D) Confirmation of Flag-Cdx2 expression in induced ES cells. Western blot and immunofluorescence analysis of Flag-Cdx2 expression at 24 hours following removal of doxycycline. (E) ChIP analysis of Brg1 and Cdx2 binding to the Oct4 promoter in Cdx2-inducible ES cells. qRT-PCR was used to determine the relative enrichment of Brg1 and Flag-Cdx2 at the Oct4 ARE versus an intergenic region in uninduced and induced ES cell extracts. A non-specific rabbit IgG was included as a negative control. Data were normalized to 1% input DNA. Asterisks denote significant differences between uninduced and induced samples (p<0.05). These experiments were replicated 3 to 4 times using two different batches of Cdx2-inducible ES cell extracts.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot, Immunofluorescence, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Schematic diagram of Oct4 regulation in blastocysts. In the trophectoderm Brg1 is recruited to the ARE of the Oct4 promoter via Cdx2. Once at the Oct4 promoter Brg1 and Cdx2 facilitate recruitment of additional co-repressors to repress transcription.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques:

Journal: bioRxiv

Article Title: Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

doi: 10.1101/349407

Figure Lengend Snippet: (A) GFP u* degradation requires catalytically active UBE3C. Top: Wild type or catalytically inactive mutant UBE3C (UBE3C C1051A ) was expressed in UBE3C KO cells and GFP u* turnover was assessed following inhibition of protein synthesis with emetine for the indicated times. Bottom: Quantification of GFP u* turnover. Data are representative of two independent experiments. (B) UBE3C is required for conjugation of high molecular weight Ub conjugates to GFP u* . Dox-induced GFP u* reporter cells were treated with the indicated inhibitors for 4 hr. PolyUb conjugates were affinity captured from cell lysates using immobilized Halo-UBQLN1 UBA and analyzed by immunoblotting with an anti-GFP antibody. (C) Conjugation of high molecular weight Ub chains to GFP u* requires catalytically active UBE3C. PolyUb conjugates were affinity purified as in (B) from wild type or UBE3C K0 cells expressing the indicated rescue constructs. Ubiquitylated GFP u* was visualized by SDS-PAGE and immunoblotting with an anti-GFP antibody. (D) Schematic diagram for the analysis in (E). Halo-TRABID NZF1 binds selectively to K29-linked Ub chains. (E) PolyUb conjugates were affinity captured from GFP u* cell lysates with immobilized Halo-TRABID NZF1 and the presence of K29 Ub linkages on GFP u* was assessed by incubating with the catalytic domain of vOTU, which does not hydrolyze K27 or K29 linkages, or with the core catalytic domain of the nonspecific DUB Usp2 (Usp2cc). Ubiquitylated species were separated by SDS-PAGE and GFP u* was visualized by immunoblotting with an anti-GFP antibody. Arrows indicate polyUb GFP u* species that are resistant to deubiquitylation by vOTU. See also .

Article Snippet: The primary antibodies used in this study are rabbit α-UBE2G2 mAb (Abcam ab174296; 1:2,500); rabbit α-AUP1 pAb (Proteintech 13726-1-AP, 1:1,000); rabbit α-UBE3C pAb (Bethyl Laboratories A304-122A, 1:1,000); mouse α-UBE2D3 mAb (Abcam ab58251; 1:2,000); mouse α-GFP JL8 mAb (Takara 632381; 1:2,000); rabbit α-GFP mAb (Cell Signaling Technology 2956; 1:1,000); mouse α-PDI mAb (Enzo Life Sciences ADI-SPA-891; 1:1000); rabbit α-VCP pAb (Novus Biologicals NB100-1558; 1:10,000); rabbit α-GAPDH pAb (Millipore ABS16; 1:20,000); mouse α-GAPDH mAb (Proteintech 60004-1-Ig; 1:20,000); rabbit α-alpha tubulin pAb (Abcam ab15246; 1:10,000); mouse α-RTA mAb (BioRad Laboratories MCA2865; 1:2,000); mouse α-CD147 mAb (Santa Cruz Biotechnology Inc. sc-25273; 1:1,000); rabbit α-SEL1L rAb (custom antibody; 1:1,000); rabbit α-Ub pAb (Cell Signaling Technology 3933S; 1:1,000); mouse α-Ub FK2 mAb (Millipore Sigma 04-263; 1:1,000); rabbit α-K48 polyUb, clone Apu2 mAb (Millipore Sigma 05-1307; 1:5,000); human α-K48 polyUb, clone Apu2.07 (Genentech; 0.2 μg/mL); human α-K11 polyUb, clone 2A3/2E6 (Genentech; 0.5 μg/mL); human α-K11/K48 bispecific polyUb (Genentech; 0.2 μg/mL); human α-K11/gD bispecific control (Genentech; 0.2 μg/mL); human α-K48/gD bispecific control (Genentech; 0.2 μg/mL).

Techniques: Mutagenesis, Inhibition, Conjugation Assay, High Molecular Weight, Western Blot, Affinity Purification, Expressing, Construct, SDS Page

Journal: bioRxiv

Article Title: Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

doi: 10.1101/349407

Figure Lengend Snippet: (Left) The glycosylated luminal ERAD substrate A1AT NHK -GFP requires ER mannosidases and glycan recognition machinery, the membrane-embedded HRD1 dislocation complex, and ER-embedded UPS components for ER exit and delivery to the proteasome, exemplifying the canonical HRD1-dependent ERAD-L degradation pathway. Substrate-specific heterogeneity in the HRD1-dependent pathway is revealed by the luminal glycosylated protein GFP-RTA E177Q , which can engage luminal recognition factors for slow delivery to the HRD1 complex or can be rapidly dislocated and degraded by bypassing upstream quality control machinery. Dislocated ERAD substrates are modified with K48 and K11 Ub linkages and targeted for destruction by cytosolic proteasomes. (Middle) The ERAD-M substrate INSIG1-GFP utilizes GP78 for ER dislocation and ubiquitylation. Efficient delivery of this highly hydrophobic substrate to cytosolic degradation machinery may be facilitated by conjugation of K11/K48 chains that increase affinity for p97/VCP and the proteasome. (Right) Efficient degradation of the ERAD-C substrate GFP u* requires conjugation of heterotypic K48/K29 chains via the ER-embedded E3 ligase TRC8 and the cytosolic E3 UBE3C, which may facilitate interactions with proteasome shuttling factors or increase substrate processing at the proteasome.

Article Snippet: The primary antibodies used in this study are rabbit α-UBE2G2 mAb (Abcam ab174296; 1:2,500); rabbit α-AUP1 pAb (Proteintech 13726-1-AP, 1:1,000); rabbit α-UBE3C pAb (Bethyl Laboratories A304-122A, 1:1,000); mouse α-UBE2D3 mAb (Abcam ab58251; 1:2,000); mouse α-GFP JL8 mAb (Takara 632381; 1:2,000); rabbit α-GFP mAb (Cell Signaling Technology 2956; 1:1,000); mouse α-PDI mAb (Enzo Life Sciences ADI-SPA-891; 1:1000); rabbit α-VCP pAb (Novus Biologicals NB100-1558; 1:10,000); rabbit α-GAPDH pAb (Millipore ABS16; 1:20,000); mouse α-GAPDH mAb (Proteintech 60004-1-Ig; 1:20,000); rabbit α-alpha tubulin pAb (Abcam ab15246; 1:10,000); mouse α-RTA mAb (BioRad Laboratories MCA2865; 1:2,000); mouse α-CD147 mAb (Santa Cruz Biotechnology Inc. sc-25273; 1:1,000); rabbit α-SEL1L rAb (custom antibody; 1:1,000); rabbit α-Ub pAb (Cell Signaling Technology 3933S; 1:1,000); mouse α-Ub FK2 mAb (Millipore Sigma 04-263; 1:1,000); rabbit α-K48 polyUb, clone Apu2 mAb (Millipore Sigma 05-1307; 1:5,000); human α-K48 polyUb, clone Apu2.07 (Genentech; 0.2 μg/mL); human α-K11 polyUb, clone 2A3/2E6 (Genentech; 0.5 μg/mL); human α-K11/K48 bispecific polyUb (Genentech; 0.2 μg/mL); human α-K11/gD bispecific control (Genentech; 0.2 μg/mL); human α-K48/gD bispecific control (Genentech; 0.2 μg/mL).

Techniques: Glycoproteomics, Membrane, Control, Modification, Conjugation Assay