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MedChemExpress wdr5 inhibitor wdr5

(A–D) Wild-type (WT) T cells were labeled with CFSE and activated in the presence of MM-401, <t>WDR5-IN-4,</t> or DMSO. After 4 days, cells were analyzed by flow cytometry to assess the effects of MM-401 (A) and <t>WDR5-IN-4</t> (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.

Wdr5 Inhibitor Wdr5, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hy+111753a/bio_rxiv__64898__2026__04__03__715913-285-24-27?v=MedChemExpress
Average 92 stars, based on 4 article reviews

wdr5 inhibitor wdr5 - by Bioz Stars, 2026-07

92/100 stars

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1) Product Images from "The MLL1–MENIN complex preserves CD8 T cell memory through a TOX–BTLA-TCF1 axis"

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

Journal: bioRxiv

doi: 10.64898/2026.04.03.715913

(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.

Figure Legend 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.

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

(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.

Figure Legend 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.

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

Image Search Results

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.

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

( 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.

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

( 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.

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.

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

( 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.

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.

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

( 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.

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.

Techniques: Western Blot, Transfection, In Vivo, Immunoprecipitation

( 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.

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.

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

( 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.

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.

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

( 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.

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.

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

( 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.

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.

Techniques: Quantitative RT-PCR, Modification, Transfection

Primers used for quantitative real-time polymerase chain reaction (qRT-PCR).

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).

Techniques: Real-time Polymerase Chain Reaction

WDR5 directly binds to the CRY-binding regions of PER1 and PER2 and increases the circadian oscillation amplitude. a Domains (yellow) and interaction partners (blue) of PER1, PER2 and WDR5. Red boxes indicate potential WDR5 binding WIN (ARART, ARPS) - and WBM (L/IDVT) motifs in PER1/2. PER1/2 have two PAS (PER-ARNT-SIM) domains (PAS-A, PAS-B) to dimerize and interact with other proteins. The middle and C-terminal regions of PER1/2 interact with CK1δ/ε and CRY1/2, respectively. WDR5 consists of 7 WD repeats (WD1-7) and has two binding sites to interact with proteins containing a WIN- or WBM motif. b Alignment of WDR5 binding motifs. WIN motifs have a conserved central arginine (bold), WBM motifs consist of a conserved aspartate (bold) flanked by aliphatic amino acids. c WDR5 overexpression increases the amplitude of circadian Bmal1-luc oscillations in human U2OS cells. Left: Bioluminescence of U2OS cells transiently transfected with the Bmal1-luc circadian reporter construct and either GFP (control) or WDR5 (n = 6 replicates from 3 independent experiments). Data are plotted as an average (colored lines) with the SEM (black lines). Middle: relative amplitude (arbitrary units), * P = 0. 035 (two-tailed unpaired t test) shows statistically significant amplitude difference between GFP and WDR5. Right: western blot of WDR5-V5 expression and GAPDH loading control in transiently transfected U2OS cells. d Analytical SEC (S75 10/300) of WDR5 23-334 and PER2 1132-1252. Top: chromatograms of WDR5 23-334 (black), PER2 1132-1252 (blue) and PER2/WDR5 complex (red). Bottom : SDS-PAGE of PER2/WDR5 complex SEC. M: molecular weight marker. The PER2/WDR5 complex elutes earlier (10 mL) than free WDR5 (> 11 mL). e Analytical SEC (S200 10/300) of WDR5 23-334 and PER1 1013-1291. Top: chromatograms of WDR5 23-334 (black), PER1 1013-1291 (blue) and PER1/WDR5 complex (red). Bottom: SDS-PAGE of Per1/WDR5 complex SEC. The PER1/WDR5 complex elutes earlier (∼13 mL) than free WDR5 (> 16 mL).

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: WDR5 directly binds to the CRY-binding regions of PER1 and PER2 and increases the circadian oscillation amplitude. a Domains (yellow) and interaction partners (blue) of PER1, PER2 and WDR5. Red boxes indicate potential WDR5 binding WIN (ARART, ARPS) - and WBM (L/IDVT) motifs in PER1/2. PER1/2 have two PAS (PER-ARNT-SIM) domains (PAS-A, PAS-B) to dimerize and interact with other proteins. The middle and C-terminal regions of PER1/2 interact with CK1δ/ε and CRY1/2, respectively. WDR5 consists of 7 WD repeats (WD1-7) and has two binding sites to interact with proteins containing a WIN- or WBM motif. b Alignment of WDR5 binding motifs. WIN motifs have a conserved central arginine (bold), WBM motifs consist of a conserved aspartate (bold) flanked by aliphatic amino acids. c WDR5 overexpression increases the amplitude of circadian Bmal1-luc oscillations in human U2OS cells. Left: Bioluminescence of U2OS cells transiently transfected with the Bmal1-luc circadian reporter construct and either GFP (control) or WDR5 (n = 6 replicates from 3 independent experiments). Data are plotted as an average (colored lines) with the SEM (black lines). Middle: relative amplitude (arbitrary units), * P = 0. 035 (two-tailed unpaired t test) shows statistically significant amplitude difference between GFP and WDR5. Right: western blot of WDR5-V5 expression and GAPDH loading control in transiently transfected U2OS cells. d Analytical SEC (S75 10/300) of WDR5 23-334 and PER2 1132-1252. Top: chromatograms of WDR5 23-334 (black), PER2 1132-1252 (blue) and PER2/WDR5 complex (red). Bottom : SDS-PAGE of PER2/WDR5 complex SEC. M: molecular weight marker. The PER2/WDR5 complex elutes earlier (10 mL) than free WDR5 (> 11 mL). e Analytical SEC (S200 10/300) of WDR5 23-334 and PER1 1013-1291. Top: chromatograms of WDR5 23-334 (black), PER1 1013-1291 (blue) and PER1/WDR5 complex (red). Bottom: SDS-PAGE of Per1/WDR5 complex SEC. The PER1/WDR5 complex elutes earlier (∼13 mL) than free WDR5 (> 16 mL).

Article Snippet: To analyze the effect of the WDR5-IN-4-TFA (C6) compound (MCE, HY-111753A, MEdChemTronica, Sweden) on circadian oscillations, U2OS cells stably infected with a lentiviral Bmal1-luciferase circadian reporter were used .

Techniques: Binding Assay, Over Expression, Transfection, Construct, Control, Two Tailed Test, Western Blot, Expressing, SDS Page, Molecular Weight, Marker

Sequence alignment of PER1 and PER2 and interaction of PER2 1132-1252 with CRY1. a Alignment of PER1 1105-1210 and PER2 1114-1219. Identical amino acids are shown by “:” and similar amino acids by “.”. The WDR5 binding motifs are shown in cyan (WBM) and yellow (WIN). Important amino acids of PER2 for interaction with CRY1 (blue) (Schmalen et al, 2014) or WDR5 (green) are highlighted as dots. Amino acids mutated within this study are indicated by arrows under (PER2) or above (PER1) the alignment. Amino acids that interact with CRY1 or with the WDR5 WBM site are mostly conserved between PER1 and PER2. The WIN motif of PER1 is not conserved in PER2. b-d A representative ITC experiment for b PER2 1132-1252 I1205G, c PER2 D1206A and d PER2 V1207A (titrant) with CRY1 1-496 (PHR) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line).

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Sequence alignment of PER1 and PER2 and interaction of PER2 1132-1252 with CRY1. a Alignment of PER1 1105-1210 and PER2 1114-1219. Identical amino acids are shown by “:” and similar amino acids by “.”. The WDR5 binding motifs are shown in cyan (WBM) and yellow (WIN). Important amino acids of PER2 for interaction with CRY1 (blue) (Schmalen et al, 2014) or WDR5 (green) are highlighted as dots. Amino acids mutated within this study are indicated by arrows under (PER2) or above (PER1) the alignment. Amino acids that interact with CRY1 or with the WDR5 WBM site are mostly conserved between PER1 and PER2. The WIN motif of PER1 is not conserved in PER2. b-d A representative ITC experiment for b PER2 1132-1252 I1205G, c PER2 D1206A and d PER2 V1207A (titrant) with CRY1 1-496 (PHR) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line).

Techniques: Sequencing, Binding Assay

The PER2 WBM motif mediates WDR5 interactions of the purified PER2-CBD protein and of full-length PER2 in HEK293 cells. a-c Crystal structure of WDR5 23-334 (green) bound to the PER2 1198-1211 WBM motif peptide (cyan). a Overview of the PER2/WDR5 complex. b Close-up view of the PER2-WDR5 interaction site. c Schematic representation of PER2-WDR5 interactions. Hydrogen bonds were defined by a maximum donor-acceptor distance of 3.1 Å, hydrophobic interactions by a maximum distance of 4.1 Å. d ITC experiment of wildtype (WT) PER2 1132-1252 (CBD) and WDR5 23-334. Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). Titrant: WDR5. e ITC results for interactions of PER2 1132-1252 (WT, I1205G, D1206A, V1207A) with WDR5 23-334 (WT, N225A) as titrant. The K D value for PER2 WT is the means +/- SD from triplicates ( , , ). f Co-IP of Flag-tagged full-length PER2 (WT, R634A, I1205G, D1206A, V1207A) with V5-tagged WDR5 (WT, N225A) in HEK293 cells. Left: relative PER2 bound to WDR5, normalized to protein expression. Error bars: +/- SEM of 3 replicates . Right: Co-IPs with anti-V5 antibody. WDR5-bound Flag-PER2 was quantified by western blot.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: The PER2 WBM motif mediates WDR5 interactions of the purified PER2-CBD protein and of full-length PER2 in HEK293 cells. a-c Crystal structure of WDR5 23-334 (green) bound to the PER2 1198-1211 WBM motif peptide (cyan). a Overview of the PER2/WDR5 complex. b Close-up view of the PER2-WDR5 interaction site. c Schematic representation of PER2-WDR5 interactions. Hydrogen bonds were defined by a maximum donor-acceptor distance of 3.1 Å, hydrophobic interactions by a maximum distance of 4.1 Å. d ITC experiment of wildtype (WT) PER2 1132-1252 (CBD) and WDR5 23-334. Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). Titrant: WDR5. e ITC results for interactions of PER2 1132-1252 (WT, I1205G, D1206A, V1207A) with WDR5 23-334 (WT, N225A) as titrant. The K D value for PER2 WT is the means +/- SD from triplicates ( , , ). f Co-IP of Flag-tagged full-length PER2 (WT, R634A, I1205G, D1206A, V1207A) with V5-tagged WDR5 (WT, N225A) in HEK293 cells. Left: relative PER2 bound to WDR5, normalized to protein expression. Error bars: +/- SEM of 3 replicates . Right: Co-IPs with anti-V5 antibody. WDR5-bound Flag-PER2 was quantified by western blot.

Techniques: Purification, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet:

Techniques:

Interaction of PER2 with WDR5. a 2Fo-Fc electron density map of the WDR5-bound PER2 WBM motif peptide (Y 1198 TGGLPTAIDVTGCV 1211 ) at 1.35σ (blue). Residues T1203 to V1211 of the PER2 peptide (cyan) are well resolved in the electron density. T1203 is modelled as alanine. WDR5 is shown in green. b Close-up view of the WBM motif of RbBP5 (orange, PDB 2XL3) and PER2 (cyan) binding to WDR5. WDR5 of the WDR5/PER2 complex is shown in green. N225 and Q289 of the WDR5/RbBP5 complex are shown in purple. The structures were superimposed on WDR5 with an RMSD of 0.271 Å c,d SEC chromatograms of WDR5 23-334 and PER2 1132-1252 (WT, I1205G, D1206A, V1207A). c Chromatograms of excess WDR5 mixed 3:1 with PER2 WT (red), PER2 D1206A (magenta) or PER2 V1207A (green). WDR5 23-334 alone (black) and PER2 1132-1252 WT alone (blue) are shown for comparison. An S75 10/300 column was used. d Chromatograms of excess WDR5 mixed 3:1 with PER2 WT (red) or PER2 I1205G (green). WDR5 23-334 alone (black) and PER2 1132-1252 WT alone (blue) are shown for comparison. An S200 10/300 column was used. e,f,g Representative ITC experiments for e PER2 1132-1252 D1206A, f V1207A and g I1205G with WDR5 23-334 as titrant are shown. The K D -value for PER2 1132-1252 V1207A (f) is the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER2 1132-1252 D1206A (e) and I1205G (g) no binding was observed.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Interaction of PER2 with WDR5. a 2Fo-Fc electron density map of the WDR5-bound PER2 WBM motif peptide (Y 1198 TGGLPTAIDVTGCV 1211 ) at 1.35σ (blue). Residues T1203 to V1211 of the PER2 peptide (cyan) are well resolved in the electron density. T1203 is modelled as alanine. WDR5 is shown in green. b Close-up view of the WBM motif of RbBP5 (orange, PDB 2XL3) and PER2 (cyan) binding to WDR5. WDR5 of the WDR5/PER2 complex is shown in green. N225 and Q289 of the WDR5/RbBP5 complex are shown in purple. The structures were superimposed on WDR5 with an RMSD of 0.271 Å c,d SEC chromatograms of WDR5 23-334 and PER2 1132-1252 (WT, I1205G, D1206A, V1207A). c Chromatograms of excess WDR5 mixed 3:1 with PER2 WT (red), PER2 D1206A (magenta) or PER2 V1207A (green). WDR5 23-334 alone (black) and PER2 1132-1252 WT alone (blue) are shown for comparison. An S75 10/300 column was used. d Chromatograms of excess WDR5 mixed 3:1 with PER2 WT (red) or PER2 I1205G (green). WDR5 23-334 alone (black) and PER2 1132-1252 WT alone (blue) are shown for comparison. An S200 10/300 column was used. e,f,g Representative ITC experiments for e PER2 1132-1252 D1206A, f V1207A and g I1205G with WDR5 23-334 as titrant are shown. The K D -value for PER2 1132-1252 V1207A (f) is the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER2 1132-1252 D1206A (e) and I1205G (g) no binding was observed.

Techniques: Binding Assay, Comparison

Impact of WDR5 N225A mutation on PER2-WDR5-, PER1-WDR5- and RbBP5-WDR5 interaction. a,c,d,e A representative ITC experiment for PER2 1132-1252 with a WDR5 23-334 N225A, c PER1 1013-1291 with WDR5 23-334 N225A, d RbBP5 with WDR5 23-334 WT, and e RbBP5 with WDR5 23-334 N225A is shown. The K D values are the means of the triplicates with standard deviations (+/- SD), also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER2 1132-1252 with WDR5 N225A (a) no binding was observed ( , ). b Close-up view of the WDR5 N225A mutant crystal structure (magenta) superimposed with the WDR5 WT structure (green, PDB 2H9L) around amino acid N225 (RMSD=0.343 Å). K227 is modeled as alanine in the WDR5 N225A structure due to conformational disorder.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Impact of WDR5 N225A mutation on PER2-WDR5-, PER1-WDR5- and RbBP5-WDR5 interaction. a,c,d,e A representative ITC experiment for PER2 1132-1252 with a WDR5 23-334 N225A, c PER1 1013-1291 with WDR5 23-334 N225A, d RbBP5 with WDR5 23-334 WT, and e RbBP5 with WDR5 23-334 N225A is shown. The K D values are the means of the triplicates with standard deviations (+/- SD), also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER2 1132-1252 with WDR5 N225A (a) no binding was observed ( , ). b Close-up view of the WDR5 N225A mutant crystal structure (magenta) superimposed with the WDR5 WT structure (green, PDB 2H9L) around amino acid N225 (RMSD=0.343 Å). K227 is modeled as alanine in the WDR5 N225A structure due to conformational disorder.

Techniques: Mutagenesis, Binding Assay

Role of PER2 WBM motif for CRY interactions and competition of WDR5 and CRY1 for PER2 binding. a ITC experiment for PER2 1132-1252 WT (CBD) with CRY1 1-496 (PHR). Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). Titrant: PER2 b Close up view of PER2 WBM motif region (cyan) bound to CRY1-PHR (red) (PDB: 4CT0, ). c ITC results (K D values) for binding of PER2 1132-1252 WT, I1205G, D1206A and V1207A (titrant) to CRY1 PHR (mean K D values +/- SD from triplicates) ( , ). d Co-IP results of Flag-tagged PER2 (WT, R634A, I1205G, D1206A, V1207A) with V5-tagged CRY1 in HEK293 cells . Left: relative PER2 bound to CRY1 normalized to protein expression. Error bars: +/- SEM of 3 replicates. Right: Co-IPs with anti-V5 antibody. CRY1-bound Flag-PER2 was quantified by western blot. e SEC analysis (S200 10/300) of WDR5 23-334, CRY1 1-496 and PER2 1132-1252. Top: chromatograms of WDR5 (black), CRY1 (magenta) and of a preformed PER2/WDR5 complex (PER2:WDR5) mixed with CRY1 (green). Bottom: SDS-PAGE of SEC run with the PER2:WDR5 + CRY1 mixture. No trimeric PER2/WDR5/CRY1 complex was observed, but only a PER2/CRY1 complex eluting at 14.3 to 15.1 mL. WDR5 is displaced by CRY1 and co-elutes with monomeric WDR5 (16.3 to 17.5 mL).

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Role of PER2 WBM motif for CRY interactions and competition of WDR5 and CRY1 for PER2 binding. a ITC experiment for PER2 1132-1252 WT (CBD) with CRY1 1-496 (PHR). Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). Titrant: PER2 b Close up view of PER2 WBM motif region (cyan) bound to CRY1-PHR (red) (PDB: 4CT0, ). c ITC results (K D values) for binding of PER2 1132-1252 WT, I1205G, D1206A and V1207A (titrant) to CRY1 PHR (mean K D values +/- SD from triplicates) ( , ). d Co-IP results of Flag-tagged PER2 (WT, R634A, I1205G, D1206A, V1207A) with V5-tagged CRY1 in HEK293 cells . Left: relative PER2 bound to CRY1 normalized to protein expression. Error bars: +/- SEM of 3 replicates. Right: Co-IPs with anti-V5 antibody. CRY1-bound Flag-PER2 was quantified by western blot. e SEC analysis (S200 10/300) of WDR5 23-334, CRY1 1-496 and PER2 1132-1252. Top: chromatograms of WDR5 (black), CRY1 (magenta) and of a preformed PER2/WDR5 complex (PER2:WDR5) mixed with CRY1 (green). Bottom: SDS-PAGE of SEC run with the PER2:WDR5 + CRY1 mixture. No trimeric PER2/WDR5/CRY1 complex was observed, but only a PER2/CRY1 complex eluting at 14.3 to 15.1 mL. WDR5 is displaced by CRY1 and co-elutes with monomeric WDR5 (16.3 to 17.5 mL).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Expressing, Western Blot, SDS Page

$PER1 predominantly binds to the WDR5 WIN site and can form a trimeric PER1/WDR5/CRY1 complex. a,b ITC analyses of the interaction of PER1 1013-1291 with a WDR5 23-334 (titrant WDR5) and with b CRY1 1-496 (PHR) (titrant PER1). Data were fit with a one-site binding model (lower panel). c ITC results (K D ) for interactions of PER1 1013-1291 (WT, R1111A, D1197A, R1111A D1197A, R1111A R1113A) with WDR5 23-334 (WT, N225A) or CRY1 1-496 (mean K D values +/- SD from triplicates) ( , and ). d SEC (S200 10/300) analyses of the interaction of PER1 1013-1291 with WDR5 23-334 WT ( top), the WIN site triple mutant WDR5 D107A F133A Y260A and the WIN/WBM site mutant WDR5 D107A F133A Y260A N225A. The SEC run of PER1 alone is shown for comparison (bottom). Left: SDS-PAGE analyses of peak SEC fractions, right: SEC chromatograms of the PER1/WDR5 complex (red), PER1 alone (blue) and WDR5 alone (black). e SEC (S200 10/300) analyses of the PER1 1013-1291 interaction with CRY1 1-496 and WDR5 23-334. Top, left : SDS-PAGE of PER1/CRY1 complex SEC, right: chromatograms of PER1 (blue), CRY1 (magenta) and PER1/CRY1 complex (orange). Middle, left: SDS-PAGE of SEC with equimolar mixture of PER1, CRY1 and WDR5, right: chromatograms of WDR5 (black), PER1/CRY1 complex (orange) and CRY1+PER1+WDR5 mixture (green). Only a dimeric PER1/CRY1 complex is observed, WDR5 is displaced. Bottom: SDS-PAGE (left) and chromatogram (right) of a preformed PER1/WDR5 complex (PER1:WDR5) incubated with equimolar CRY1 (green). A trimeric PER1/WDR5/CRY1 complex is formed.$

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: PER1 predominantly binds to the WDR5 WIN site and can form a trimeric PER1/WDR5/CRY1 complex. a,b ITC analyses of the interaction of PER1 1013-1291 with a WDR5 23-334 (titrant WDR5) and with b CRY1 1-496 (PHR) (titrant PER1). Data were fit with a one-site binding model (lower panel). c ITC results (K D ) for interactions of PER1 1013-1291 (WT, R1111A, D1197A, R1111A D1197A, R1111A R1113A) with WDR5 23-334 (WT, N225A) or CRY1 1-496 (mean K D values +/- SD from triplicates) ( , and ). d SEC (S200 10/300) analyses of the interaction of PER1 1013-1291 with WDR5 23-334 WT ( top), the WIN site triple mutant WDR5 D107A F133A Y260A and the WIN/WBM site mutant WDR5 D107A F133A Y260A N225A. The SEC run of PER1 alone is shown for comparison (bottom). Left: SDS-PAGE analyses of peak SEC fractions, right: SEC chromatograms of the PER1/WDR5 complex (red), PER1 alone (blue) and WDR5 alone (black). e SEC (S200 10/300) analyses of the PER1 1013-1291 interaction with CRY1 1-496 and WDR5 23-334. Top, left : SDS-PAGE of PER1/CRY1 complex SEC, right: chromatograms of PER1 (blue), CRY1 (magenta) and PER1/CRY1 complex (orange). Middle, left: SDS-PAGE of SEC with equimolar mixture of PER1, CRY1 and WDR5, right: chromatograms of WDR5 (black), PER1/CRY1 complex (orange) and CRY1+PER1+WDR5 mixture (green). Only a dimeric PER1/CRY1 complex is observed, WDR5 is displaced. Bottom: SDS-PAGE (left) and chromatogram (right) of a preformed PER1/WDR5 complex (PER1:WDR5) incubated with equimolar CRY1 (green). A trimeric PER1/WDR5/CRY1 complex is formed.

Techniques: Binding Assay, Mutagenesis, Comparison, SDS Page, Incubation

Interaction of PER1 1013-1291 with WDR5. a-d A representative ITC experiment for a PER1 1013-1291 D1197A, b PER1 R1111A, c PER1 R1111A D1197A and d PER1 R1111A R1113A with WDR5 23-334 (titrant) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER1 R1111A R1113A and WDR5 (d) only very weak binding was observed, with an estimated K D > 100 µM. e Overview of the structure of WDR5 (green) bound to a KANSL1 WIN motif peptide (cyan) in the WIN motif binding pocket and to a KANSL2 WBM motif peptide (purple) in the WBM motif binding pocket of WDR5 (PDB 4CY2) . Important amino acids in the WIN motif binding pocket of WDR5 (D107, F133, Y260) are shown in pink. The two WIN motif arginine residues of KANSL1 (denoted R1-WIN, R3-WIN) correspond to R1111 (R1) and R1113 (R3) of PER1. N225 in the WBM motif binding pocket of WDR5 is highlighted in orange. The conserved aspartate (denoted D-WBM) in the KANSL2 WBM motif peptide corresponds to D1197 in PER1 and D1206 in PER2.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Interaction of PER1 1013-1291 with WDR5. a-d A representative ITC experiment for a PER1 1013-1291 D1197A, b PER1 R1111A, c PER1 R1111A D1197A and d PER1 R1111A R1113A with WDR5 23-334 (titrant) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). For PER1 R1111A R1113A and WDR5 (d) only very weak binding was observed, with an estimated K D > 100 µM. e Overview of the structure of WDR5 (green) bound to a KANSL1 WIN motif peptide (cyan) in the WIN motif binding pocket and to a KANSL2 WBM motif peptide (purple) in the WBM motif binding pocket of WDR5 (PDB 4CY2) . Important amino acids in the WIN motif binding pocket of WDR5 (D107, F133, Y260) are shown in pink. The two WIN motif arginine residues of KANSL1 (denoted R1-WIN, R3-WIN) correspond to R1111 (R1) and R1113 (R3) of PER1. N225 in the WBM motif binding pocket of WDR5 is highlighted in orange. The conserved aspartate (denoted D-WBM) in the KANSL2 WBM motif peptide corresponds to D1197 in PER1 and D1206 in PER2.

Techniques: Binding Assay

Interaction of PER1 1013-1291 with CRY1 and CRY2. a-c A representative ITC experiment for a PER1 1013-1291 R1111A, b PER1 D1197A and c PER1 R1111A D1197A (titrant) with CRY1 1-496 (PHR) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). d Chromatograms (left) and SDS-PAGE analyses (right) of SEC (S200 10/300) of the PER1 1013-1291 interaction with CRY2 1-512 (PHR) and WDR5 23-334. SDS-PAGE top: PER1/WDR5 complex (red chromatogram); second: preformed PER1/WDR5 complex incubated with equimolar CRY2 (light green chromatogram). A trimeric PER1/WDR5/CRY2 complex is formed, that starts to elute at 11.1 mL; third: equimolar mixture of PER1, CRY2 and WDR5 (dark green chromatogram). Only a dimeric PER1/CRY2 complex is observed, WDR5 is displaced; bottom: PER1/CRY2 complex (orange chromatogram), that starts to elute at 11.6 mL, i.e. later than the trimeric PER1/WDR5/CRY2 complex.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Interaction of PER1 1013-1291 with CRY1 and CRY2. a-c A representative ITC experiment for a PER1 1013-1291 R1111A, b PER1 D1197A and c PER1 R1111A D1197A (titrant) with CRY1 1-496 (PHR) is shown. The K D values are the means of the triplicates +/- SD, also shown in and . Upper panel : raw data, lower panel : integrated heat (points) with fit for a one-site binding model (line). d Chromatograms (left) and SDS-PAGE analyses (right) of SEC (S200 10/300) of the PER1 1013-1291 interaction with CRY2 1-512 (PHR) and WDR5 23-334. SDS-PAGE top: PER1/WDR5 complex (red chromatogram); second: preformed PER1/WDR5 complex incubated with equimolar CRY2 (light green chromatogram). A trimeric PER1/WDR5/CRY2 complex is formed, that starts to elute at 11.1 mL; third: equimolar mixture of PER1, CRY2 and WDR5 (dark green chromatogram). Only a dimeric PER1/CRY2 complex is observed, WDR5 is displaced; bottom: PER1/CRY2 complex (orange chromatogram), that starts to elute at 11.6 mL, i.e. later than the trimeric PER1/WDR5/CRY2 complex.

Techniques: Binding Assay, SDS Page, Incubation

Effect of WIN- and WBM interface mutations and C6 WIN site inhibitor on PER1-WDR5- and PER1-CRY1 interactions in HEK293 cells and on circadian oscillations in U2OS cells a Co-IP of Flag-tagged full-length PER1 (WT, R1111A R1113A, D1197A, R1111A D1197A) with V5-tagged WDR5 (WT, N225A) in HEK293 cells. The C6 compound was added to wildtype PER1 and WDR5 at a 5 µM concentration. Left: R elative PER1 bound to WDR5 normalized to protein expression. Right: Co-IPs with anti-V5 antibody. WDR5-bound Flag-PER1 was quantified by western blot. b Co-IP of Flag-tagged PER1 (WT, R1111A R1113A, D1197A, R1111A D1197A) with V5-tagged CRY1 in HEK293 cells. Left: Relative PER1 bound to CRY1 normalized to protein expression . Right: Co-IPs with anti-V5 antibody. CRY1-bound Flag-PER1 was quantified by western blot. Error bars in a,b: +/- SEM of 3 replicates. c Chemical structure of the C6 WIN site inhibitor . d C6 shortens the period of circadian Bmal1-Luc oscillations in U2OS cells by 2 to 3 h at all tested concentrations but does not significantly change the oscillation amplitude. Left: average relative bioluminescence n=3, middle: Period length, * p<0.05, ** p<0.01, two-tailed unpaired t test, right: relative amplitude (arbitrary units), no significant differences, two-tailed unpaired t test.

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Effect of WIN- and WBM interface mutations and C6 WIN site inhibitor on PER1-WDR5- and PER1-CRY1 interactions in HEK293 cells and on circadian oscillations in U2OS cells a Co-IP of Flag-tagged full-length PER1 (WT, R1111A R1113A, D1197A, R1111A D1197A) with V5-tagged WDR5 (WT, N225A) in HEK293 cells. The C6 compound was added to wildtype PER1 and WDR5 at a 5 µM concentration. Left: R elative PER1 bound to WDR5 normalized to protein expression. Right: Co-IPs with anti-V5 antibody. WDR5-bound Flag-PER1 was quantified by western blot. b Co-IP of Flag-tagged PER1 (WT, R1111A R1113A, D1197A, R1111A D1197A) with V5-tagged CRY1 in HEK293 cells. Left: Relative PER1 bound to CRY1 normalized to protein expression . Right: Co-IPs with anti-V5 antibody. CRY1-bound Flag-PER1 was quantified by western blot. Error bars in a,b: +/- SEM of 3 replicates. c Chemical structure of the C6 WIN site inhibitor . d C6 shortens the period of circadian Bmal1-Luc oscillations in U2OS cells by 2 to 3 h at all tested concentrations but does not significantly change the oscillation amplitude. Left: average relative bioluminescence n=3, middle: Period length, * p<0.05, ** p<0.01, two-tailed unpaired t test, right: relative amplitude (arbitrary units), no significant differences, two-tailed unpaired t test.

Techniques: Co-Immunoprecipitation Assay, Concentration Assay, Expressing, Western Blot, Two Tailed Test

$Interaction interplay between PER, WDR5, RbBP5, MLL1 and CRY. a Analytical SEC (S200 10/300) of PER1 1013-1291, PER2 1132-1252 and WDR5 23-334. Chromatogram (top) and SDS-PAGE (bottom) of the SEC run with the PER1+WDR5+PER2 mixture. No trimeric PER1/WDR5/PER2 complex was observed, but only dimeric PER1/WDR5 and PER2/WDR5 complexes. Arrows indicate elution volumes of individual PER1/WDR5- and PER2/WDR5 complexes and of WDR5. b Analytical SEC (S200 10/300) of PER2 1132-1252, RbBP5 and WDR5 23-334. Top: chromatograms of PER2/WDR5 (red)- and RbBP5/WDR5 (magenta) complexes (excess WDR5) and of a competition experiment of a preformed PER2/WDR5 complex (PER2:WDR5) with added equimolar RbBP5 (brown). Bottom: SDS-PAGE of the competition experiment (SEC peak fractions). No trimeric PER2/WDR5/RbBP5 complex was observed, but only the dimeric WDR5/RbBP5 complex. PER2 is displaced by RbBP5. c Analytical SEC (S200 10/300) of PER1 1013-1291, RbBP5 and WDR5 23-334. Top: chromatograms of PER1/WDR5 (red)-, RbBP5/WDR5 (magenta) and PER1/WDR5/RbBP5 (brown) complexes. Bottom: SDS-PAGE of the trimeric PER1/WDR5/RbBP5 complex (SEC peak fractions), that eluted significantly earlier than the dimeric complexes and the individual PER1 and WDR5 proteins . Note that a different SEC S200 10/300 column was used in and than in all other experiments stating S200 10/300. d Analytical SEC (S200 10/300) of WDR5 23-334, PER1 1013-1291 and hMLL1 3754-3963 (MLL1-WIN-SET). Top: chromatograms of PER1/WDR5 (red)- and MLL1/WDR5 (orange) complexes and of the competition experiment of a preformed MLL1/WDR5 complex (MLL1:WDR5) with added equimolar PER1 (black). Bottom: SDS-PAGE of the competition experiment. Newly formed PER1/WDR5 complex elutes at ∼ 12.5 mL, remaining MLL1/WDR5 complex at ∼ 15 mL. PER1 partially displaces MLL1 (∼ 16.5 mL) from the preformed MLL1/WDR5 complex. e Schematic of PER-WDR5 interactions and their interplay with CRY, RbBP5 and MLL1. PER2 only binds to the WBM site of WDR5 and another protein could bind to the WDR5 WIN site. PER1 binds to both WDR5 binding sites via its WIN- and WBM motifs, but the WIN site interaction is more important. Alternatively, PER1 can only bind to the WIN site, while another protein binds to the WBM site, for example RbBP5. Thereby PER1 could replace MLL1 on the WIN site. PER2 binds to either WDR5 or CRY, whereas PER1 can form a trimeric PER1/WDR5/CRY complex. f Active-, early repressed- and late repressed states of BMAL1/CLOCK in the mammalian circadian oscillator. WDR5 is an MLL1 complex component associated with active BMAL1/CLOCK, but was also identified in a repressive PER/CRY containing complex. PER1/WDR5/RbBP5- and PER1/WDR5/CRY complexes may be formed at the transition from the active-to the early repressed state. PER2/WDR5 complexes occur in a CRY-independent manner.$

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Interaction interplay between PER, WDR5, RbBP5, MLL1 and CRY. a Analytical SEC (S200 10/300) of PER1 1013-1291, PER2 1132-1252 and WDR5 23-334. Chromatogram (top) and SDS-PAGE (bottom) of the SEC run with the PER1+WDR5+PER2 mixture. No trimeric PER1/WDR5/PER2 complex was observed, but only dimeric PER1/WDR5 and PER2/WDR5 complexes. Arrows indicate elution volumes of individual PER1/WDR5- and PER2/WDR5 complexes and of WDR5. b Analytical SEC (S200 10/300) of PER2 1132-1252, RbBP5 and WDR5 23-334. Top: chromatograms of PER2/WDR5 (red)- and RbBP5/WDR5 (magenta) complexes (excess WDR5) and of a competition experiment of a preformed PER2/WDR5 complex (PER2:WDR5) with added equimolar RbBP5 (brown). Bottom: SDS-PAGE of the competition experiment (SEC peak fractions). No trimeric PER2/WDR5/RbBP5 complex was observed, but only the dimeric WDR5/RbBP5 complex. PER2 is displaced by RbBP5. c Analytical SEC (S200 10/300) of PER1 1013-1291, RbBP5 and WDR5 23-334. Top: chromatograms of PER1/WDR5 (red)-, RbBP5/WDR5 (magenta) and PER1/WDR5/RbBP5 (brown) complexes. Bottom: SDS-PAGE of the trimeric PER1/WDR5/RbBP5 complex (SEC peak fractions), that eluted significantly earlier than the dimeric complexes and the individual PER1 and WDR5 proteins . Note that a different SEC S200 10/300 column was used in and than in all other experiments stating S200 10/300. d Analytical SEC (S200 10/300) of WDR5 23-334, PER1 1013-1291 and hMLL1 3754-3963 (MLL1-WIN-SET). Top: chromatograms of PER1/WDR5 (red)- and MLL1/WDR5 (orange) complexes and of the competition experiment of a preformed MLL1/WDR5 complex (MLL1:WDR5) with added equimolar PER1 (black). Bottom: SDS-PAGE of the competition experiment. Newly formed PER1/WDR5 complex elutes at ∼ 12.5 mL, remaining MLL1/WDR5 complex at ∼ 15 mL. PER1 partially displaces MLL1 (∼ 16.5 mL) from the preformed MLL1/WDR5 complex. e Schematic of PER-WDR5 interactions and their interplay with CRY, RbBP5 and MLL1. PER2 only binds to the WBM site of WDR5 and another protein could bind to the WDR5 WIN site. PER1 binds to both WDR5 binding sites via its WIN- and WBM motifs, but the WIN site interaction is more important. Alternatively, PER1 can only bind to the WIN site, while another protein binds to the WBM site, for example RbBP5. Thereby PER1 could replace MLL1 on the WIN site. PER2 binds to either WDR5 or CRY, whereas PER1 can form a trimeric PER1/WDR5/CRY complex. f Active-, early repressed- and late repressed states of BMAL1/CLOCK in the mammalian circadian oscillator. WDR5 is an MLL1 complex component associated with active BMAL1/CLOCK, but was also identified in a repressive PER/CRY containing complex. PER1/WDR5/RbBP5- and PER1/WDR5/CRY complexes may be formed at the transition from the active-to the early repressed state. PER2/WDR5 complexes occur in a CRY-independent manner.

Techniques: SDS Page, Binding Assay

Interaction interplay of PER1 1013-1291 and MLL1 3754-3963 on WDR5. a Chromatograms (left) and SDS-PAGE analyses (right) of the SEC (S200 10/300) runs with the individual proteins PER1 1013-1291 (top), WDR5 23-334 (middle) and MLL1 3754 - 3966 (WIN motif + SET domain) (bottom). As MLL1 has a very low absorbance at 280 nm, we also show the chromatogram for absorbance at 215 nm. b Chromatograms (left) and SDS-PAGE analyses (right) of SEC (S200 10/300) runs with dimeric PER1/WDR5 (middle) and MLL1/WDR5 (bottom) complexes. Top : preformed MLL1/WDR5 complex incubated with equimolar PER1 (black chromatogram). PER1 partially displaces MLL1 from the preformed MLL1/WDR5 complex (identical to , added for direct comparison with SEC runs of dimeric PER1/WDR5- and MLL1/WDR5 complexes and individual PER1, MLL1 and WDR5 proteins)

Journal: bioRxiv

Article Title: A structural competition involving WDR5 times circadian oscillations

doi: 10.1101/2023.06.05.543739

Figure Lengend Snippet: Interaction interplay of PER1 1013-1291 and MLL1 3754-3963 on WDR5. a Chromatograms (left) and SDS-PAGE analyses (right) of the SEC (S200 10/300) runs with the individual proteins PER1 1013-1291 (top), WDR5 23-334 (middle) and MLL1 3754 - 3966 (WIN motif + SET domain) (bottom). As MLL1 has a very low absorbance at 280 nm, we also show the chromatogram for absorbance at 215 nm. b Chromatograms (left) and SDS-PAGE analyses (right) of SEC (S200 10/300) runs with dimeric PER1/WDR5 (middle) and MLL1/WDR5 (bottom) complexes. Top : preformed MLL1/WDR5 complex incubated with equimolar PER1 (black chromatogram). PER1 partially displaces MLL1 from the preformed MLL1/WDR5 complex (identical to , added for direct comparison with SEC runs of dimeric PER1/WDR5- and MLL1/WDR5 complexes and individual PER1, MLL1 and WDR5 proteins)

Techniques: SDS Page, Incubation, Comparison

Review

Structured Review

Images

1) Product Images from "The MLL1–MENIN complex preserves CD8 T cell memory through a TOX–BTLA-TCF1 axis"

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