wdr5 in 4  (TargetMol)

Journal: bioRxiv

Article Title: The MLL1–MENIN complex preserves CD8 T cell memory through a TOX–BTLA-TCF1 axis

doi: 10.64898/2026.04.03.715913

Figure Lengend Snippet: (A–D) Wild-type (WT) T cells were labeled with CFSE and activated in the presence of MM-401, WDR5-IN-4, or DMSO. After 4 days, cells were analyzed by flow cytometry to assess the effects of MM-401 (A) and WDR5-IN-4 (B) on cell division (CFSE dilution), and by RT-PCR to determine the effects of these inhibitors on Sell (C) and Tcf7 (D) transcription. Data are representative of two independent experiments. (E, F) Naïve CD8⁺ T cells were isolated from WT mice and activated in vitro for 4 days to generate activated T cells. Naïve and activated T cells were compared for Tcf7 expression by RT-PCR (E) and for H3K4me3 enrichment at the Tcf7 locus by ChIP-PCR (F). Data are representative of two independent experiments. (G, H) Mll1KO and WT T cells were activated in vitro and analyzed after 4 days for Tcf7 transcription by RT-PCR (G) and H3K4me3 enrichment at the Tcf7 locus by ChIP-PCR (H). Data are representative of three independent experiments. (I, J) Thymocytes and B cells were isolated from WT mice and compared for Tcf7 transcription by RT-PCR (I) and H3K4me3 enrichment at the Tcf7 locus by ChIP-PCR (J). Data are representative of two independent experiments.

Article Snippet: Small-molecule inhibitors used included AKT inhibitor MK-2206 (selleckchem) used at 0.05uM, AKT inhibitor AKTi-1/2 (selleckchem) used at 0.5uM, Menin inhibitor MI-3454 used at 0.25uM, Wdr5 inhibitor WDR5-IN-4 (Medchemexpress) used at 2.5uM, Wdr5 inhibitor MM-401 (invivochem) used at 25uM, Thymidine (for S-phase arrest) used at 2mM (sigma-aldrich), Nocodazole (for mitotic arrest) used at 0.5uM (selleckchem).

Techniques: Labeling, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Isolation, In Vitro, Expressing

Journal: bioRxiv

Article Title: The MLL1–MENIN complex preserves CD8 T cell memory through a TOX–BTLA-TCF1 axis

doi: 10.64898/2026.04.03.715913

Figure Lengend Snippet: (A) Wild-type (WT) T cells were activated in the presence of WDR5-IN-4, MM-401, or DMSO. After 4 days, cells were collected and analyzed for Tox expression by RT-PCR. Data are representative of two independent experiments. (B, E) T cells from Mll1KO mice and their WT littermates were activated in vitro. After 4 days, cells were collected and analyzed for H3K4me3 (B) and H4K16ac (E) enrichment at the Tox locus by ChIP-PCR. Data are representative of three independent experiments. (C, D, F) Thymocytes and B cells were isolated from WT mice and compared for Tox expression by RT-PCR (C), and for H3K4me3 (D) and H4K16ac (F) enrichment at the Tox locus by ChIP-PCR. Data are representative of two independent experiments. (G, H) T cells from Mll1KO mice and their WT littermates were activated in the presence of MI-3454 or DMSO. After 4 days, cells were collected and analyzed for Tox (G) and Btla (H) expression by RT-PCR. Data are representative of two independent experiments.

Article Snippet: Small-molecule inhibitors used included AKT inhibitor MK-2206 (selleckchem) used at 0.05uM, AKT inhibitor AKTi-1/2 (selleckchem) used at 0.5uM, Menin inhibitor MI-3454 used at 0.25uM, Wdr5 inhibitor WDR5-IN-4 (Medchemexpress) used at 2.5uM, Wdr5 inhibitor MM-401 (invivochem) used at 25uM, Thymidine (for S-phase arrest) used at 2mM (sigma-aldrich), Nocodazole (for mitotic arrest) used at 0.5uM (selleckchem).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Isolation

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Growth curves of SU-DIPGXIII cells transduced with shRNAs to knockdown both KAT2A and KAT2B, showing significantly reduced growth compared to control shRNA-transduced cells. b, Number of live SU-DIPGXIII+Cas9 cells expressing a control plasmid (HA/flag-GFP) or sgRNAs to KO SAGA-associated acetyltransferase activity ( TADA2B sgRNAs), or de-ubiquitinase activity ( ENY2 sgRNA). c, Schematic illustrating inhibition of histone-modifying modules of SAGA/ATAC complexes using chemical probes to target SAGA/ATAC-dependent histone acetylation (GSK4027, CPTH2, garcinol), SAGA-dependent H2A/H2B de-ubiquitination (USP22si-02), or WDR5-mediated H3K4me3 methylation (OICR-9529, WM856, WDR5-IN-4, and WDR5-IN-6). d, Average normalized cell viability from CellTiter-Glo assays conducted seven days after starting treatment of H3K27M mutant cells (teal curves) or non-transformed, H3 wildtype, control cells (black curves) with increasing doses of the KAT2A/2B bromodomain-targeting acetyltransferase inhibitor, GSK4027. e, Immunoblots confirming that treatment of BT245 cells with GSK4027 reduced H3K9ac in a dose-dependent manner, but not total H3 or SGF29 protein abundance. f-h, Dose response curves showing inhibition of H3K27M mutant DMG cell viability, but no effect on control H3 wild-type cells (black curves) following seven days of treatment with the DUB inhibitor, USP22i-S02 ( f, yellow curves), the WDR5 WIN site inhibitor, WDR5-IN-4 ( g, blue curves), or the WDR5 WBM site inhibitor, WDR5-IN-6 ( h, blue curves). i-j Average number of live SU-DIPGXIIIP*+ZsG/luc cells ( i ), and average BT245 ( j ) cell viability (CellTiter-Glo) following treatment with combinations of GSK4027 and USP22si-02 to simultaneously target both SAGA/ATAC-dependent histone acetyltransferase activity and SAGA-dependent H2A/H2B de-ubiquitination. k-l, BLISS synergy plots showing a combined effect of GSK4027 and WDR5-IN-6 treatment in reducing SU-DIPGXIIIP*+ZsG/luc growth ( k, **p<1.74×10 -3 ), and a combined effect of GSK4027 and WDR5-IN-4 treatment in reducing BT245 cell growth ( l , ****p<2.5×10 -10 ).

Article Snippet: The following compounds were used in this study: SGF29-IN-1 (cat# HY-158009, MedChemExpress), GSK4027 (cat# SML2018, Sigma Aldrich), CPTH2 (cat# J65939LB0, ThermoFisher), garcinol (cat# 14076, Active Motif), USP22si-02 (cat# SML3875, Sigma Aldrich), WDR5-IN-4 (WIN site inhibitor, cat# HY-111753, MedChemExpress), WDR5-IN-6 (WBM site inhibitor, cat# T77495, TargetMol), OICR9429 (cat# T6916, TargetMol), WM586 (WDR5/Myc inhibitor, cat# HY-153728, MedChemExpress), YM-53601 (cat# HY-100313A, MedChemExpress), NB-598 (cat# HY-16343, MedChemExpress), terbinafine (cat# T3677, TCI Chemicals), Atorvastatin (cat# SML3030, Sigma Aldrich), and Pitavastatin (cat# AMBH2D6EF677, Ambeed)

Techniques: Transduction, Knockdown, Control, shRNA, Expressing, Plasmid Preparation, Activity Assay, Inhibition, Ubiquitin Proteomics, Methylation, Mutagenesis, Transformation Assay, Western Blot, Quantitative Proteomics

Journal: bioRxiv

Article Title: SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

doi: 10.64898/2026.01.22.701194

Figure Lengend Snippet: a, Growth curves of SU-DIPGXIII cells transduced with shRNAs to knockdown both KAT2A and KAT2B, showing significantly reduced growth compared to control shRNA-transduced cells. b, Number of live SU-DIPGXIII+Cas9 cells expressing a control plasmid (HA/flag-GFP) or sgRNAs to KO SAGA-associated acetyltransferase activity ( TADA2B sgRNAs), or de-ubiquitinase activity ( ENY2 sgRNA). c, Schematic illustrating inhibition of histone-modifying modules of SAGA/ATAC complexes using chemical probes to target SAGA/ATAC-dependent histone acetylation (GSK4027, CPTH2, garcinol), SAGA-dependent H2A/H2B de-ubiquitination (USP22si-02), or WDR5-mediated H3K4me3 methylation (OICR-9529, WM856, WDR5-IN-4, and WDR5-IN-6). d, Average normalized cell viability from CellTiter-Glo assays conducted seven days after starting treatment of H3K27M mutant cells (teal curves) or non-transformed, H3 wildtype, control cells (black curves) with increasing doses of the KAT2A/2B bromodomain-targeting acetyltransferase inhibitor, GSK4027. e, Immunoblots confirming that treatment of BT245 cells with GSK4027 reduced H3K9ac in a dose-dependent manner, but not total H3 or SGF29 protein abundance. f-h, Dose response curves showing inhibition of H3K27M mutant DMG cell viability, but no effect on control H3 wild-type cells (black curves) following seven days of treatment with the DUB inhibitor, USP22i-S02 ( f, yellow curves), the WDR5 WIN site inhibitor, WDR5-IN-4 ( g, blue curves), or the WDR5 WBM site inhibitor, WDR5-IN-6 ( h, blue curves). i-j Average number of live SU-DIPGXIIIP*+ZsG/luc cells ( i ), and average BT245 ( j ) cell viability (CellTiter-Glo) following treatment with combinations of GSK4027 and USP22si-02 to simultaneously target both SAGA/ATAC-dependent histone acetyltransferase activity and SAGA-dependent H2A/H2B de-ubiquitination. k-l, BLISS synergy plots showing a combined effect of GSK4027 and WDR5-IN-6 treatment in reducing SU-DIPGXIIIP*+ZsG/luc growth ( k, **p<1.74×10 -3 ), and a combined effect of GSK4027 and WDR5-IN-4 treatment in reducing BT245 cell growth ( l , ****p<2.5×10 -10 ).

Article Snippet: The following compounds were used in this study: SGF29-IN-1 (cat# HY-158009, MedChemExpress), GSK4027 (cat# SML2018, Sigma Aldrich), CPTH2 (cat# J65939LB0, ThermoFisher), garcinol (cat# 14076, Active Motif), USP22si-02 (cat# SML3875, Sigma Aldrich), WDR5-IN-4 (WIN site inhibitor, cat# HY-111753, MedChemExpress), WDR5-IN-6 (WBM site inhibitor, cat# T77495, TargetMol), OICR9429 (cat# T6916, TargetMol), WM586 (WDR5/Myc inhibitor, cat# HY-153728, MedChemExpress), YM-53601 (cat# HY-100313A, MedChemExpress), NB-598 (cat# HY-16343, MedChemExpress), terbinafine (cat# T3677, TCI Chemicals), Atorvastatin (cat# SML3030, Sigma Aldrich), and Pitavastatin (cat# AMBH2D6EF677, Ambeed)

Techniques: Transduction, Knockdown, Control, shRNA, Expressing, Plasmid Preparation, Activity Assay, Inhibition, Ubiquitin Proteomics, Methylation, Mutagenesis, Transformation Assay, Western Blot, Quantitative Proteomics

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A ) Western blot analysis of protein levels of core components of PRC1 and PRC2 complexes in hindbrain of WT, Rnf220 +/- , and Rnf220 -/- mouse embryos at E18.5. ( B ) Western blot analysis of protein levels of core components of PRC1 and PRC2 complexes in pons of adult Rnf220 +/- and WT mice. ( C, D ) Western blot analysis of protein levels of WDR5 in cerebellum and cortex of adult Rnf220 +/- and WT mice. WT, wild-type; HE, heterozygote; KO, knockout; IB, immunoblot.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Western Blot, Knock-Out

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A–D ) Western blots analysis showing the protein level of WDR5 in the indicated brain tissues of mice with different genotypes at different ages. ( E ) Western blot analysis showing WDR5 levels in the pons of adult mice with indicated genotypes. ( F ) Western blot analysis of protein levels of WDR5 in P19 cells with Rnf220 knockdown or not in the presence or absence of RA. IB, immunoblot; WT, wild-type; HE, heterozygote; KO, knockout; PN, pontine nuclei; NC, negative control; RA, retinoic acid.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Western Blot, Knockdown, Knock-Out, Negative Control

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A–B ) Co-immunoprecipitation (co-IP) analysis of interactions between RNF220 and WDR5 in HEK293 cells. HEK293 cells were transfected with indicated plasmids and harvested after 48 hr. Cell lysates were immunoprecipitated with anti-FLAG beads. Whole-cell lysate and immunoprecipitates were subjected to western blot analysis using indicated antibodies. ( C ) Endogenous co-immunoprecipitation analysis showing the interaction between RNF220 and WDR5 in hindbrains of WT mice. ( D ) Western blots analysis shows the protein level of WDR5 when co-expressed with wild-type or mutated RNF220 in HEK293 cells. ( E ) Western blots analysis shows the protein level of WDR5 when co-expressed with RNF220 in HEK293 cells in the presence of MG132 (10 mM) or not. ( F ) In vivo ubiquitination assays showing the ubiquitination status of WDR5 when co-expressed with WT or mutated RNF220 in HEK293 cells. ( G ) In vivo ubiquitination assays showing the ubiquitination status of WDR5 in hindbrains of WT and Rnf220 +/- mice. ( H ) In vivo ubiquitination assays showing RNF220-induced polyubiquitination of WDR5 when the indicated ubiquitin mutations were used in HEK293 cells. ( I ) In vivo ubiquitination assays showing the ubiquitination status of the indicated WDR5 mutants when co-expressed with WT or ligase-dead RNF220 in HEK293 cells. WT, wild-type; HE, heterozygote; KO, knockout; IB, immunoblot; IP, immunoprecipitation; UB, ubiquitin; WCL, whole-cell lysate; △Ring, RNF220 Ring domain deletion; W539R, RNF220 ligase dead mutation; K48, ubiquitin with all lysines except the K48 mutated to arginine; K48R, ubiquitin with the K48 was substituted by an arginine; 3KR, substitution of lysines at the positions of 109, 112, and 120 in WDR5 with arginines simultaneously.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Western Blot, In Vivo, Knock-Out, Mutagenesis

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A ) Western blot analysis of the levels of three WDR5 truncated proteins in HEK293 cells when co-transfected with RNF220 or not. ( B ) In vivo ubiquitination analysis of ubiquitination status of indicated WDR5 KR mutants when co-expressed with RNF220 or not in HEK293 cells. IB, immunoblot; IP, immunoprecipitation; WCL, whole-cell lysate; K31R, K52R, K109R, K112R, K120R, K123R, or K126R, substitution of lysine with arginine in WDR5 at indicated positions.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Western Blot, Transfection, In Vivo, Immunoprecipitation

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A–B ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the expression levels of Rnf220 ( A ) and Wdr5 ( B ) when transfected the indicated combinations of small interfering RNAs (siRNAs) against Rnf220 or Wdr5 in the presence or absence of RA. Bar graphs show the relative levels normalized against control group without siRNA or RA treatment. ( C ) qRT-PCR analysis showing the expression levels of Hoxa1, Hoxb1, Hoxa9, Hoxb9 when siRnf220, together with siWDR5 or not, were transfected in P19 cells treated with RA. RA, retinoic acid. n.s., not significant; ***p<0.001.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Control

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the expression levels of Hoxa1 , Hoxb1 , Hoxa9 , Hoxb9 when transfected si Rnf220 or both si Rnf220 and si Wdr5 without RA treatment. ( B ) qRT-PCR analysis of mRNA levels of Wdr5 , Hoxa1 , Hoxb1 , Hoxa9 , and Hoxb9 when Wdr5 was knocked down by small interfering RNA (siRNA) transfection in P19 cells with or without RA treatment. Bar graphs show the relative levels normalized against control group without siRNA or RA treatment. RA, retinoic acid. n.s., not significant; *p<0.05.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Small Interfering RNA, Control

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A ) Diagram of experimental strategy for in utero local injection of WDR5 inhibitors. ( B–C ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of expression levels of Rnf220 ( B ), Hox3-Hox5 ( C ) in hindbrains of WT and Rnf220 +/- mouse embryos treated with WDR5 inhibitors or not at E18.5 (n=3 mice per group). Actin was used as the internal controls ( C ). Heatmap of Hox expression showed the relative levels normalized against WT group. ( D–E ) qRT-PCR analysis of expression levels of Rnf220 ( D ), Wdr5 ( D ), Hox3-Hox5 ( E ) in pons of P15 mice with indicated genotypes (n=2 mice per group). Gapdh was used as the internal controls ( E ). Heatmap of Hox expression showed the relative levels normalized against WT group. WT, wild-type; HE, heterozygote.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: In Utero, Injection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: ( A–B ) ChIP-qRT-PCR analysis of repressive epigenetic modification (H3K27me3) ( A ) and activated epigenetic modification (H3K4me3) ( B ) levels in promoter regions of indicated Hox genes in P19 cell line transfected with si Rnf220 or both si Rnf220 and si Wdr5 . n.s., not significant; *p<0.05, **p<0.01.

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Quantitative RT-PCR, Modification, Transfection

Journal: eLife

Article Title: The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

doi: 10.7554/eLife.94657

Figure Lengend Snippet: Primers used for quantitative real-time polymerase chain reaction (qRT-PCR).

Article Snippet: WDR5-IN-4 (100 μg, MedChemExpress, HY-111753A), containing 0.05% Malachite Green reagent for tracing, was injected into the hindbrain of E15.5 embryos using a finely tapered borosilicate needle.

Techniques: Real-time Polymerase Chain Reaction