Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation decreases methyltransferase activity and intrinsic protein stability of SETD2 catalytic core in vitro . A , upper panel : schematic representation of the SETD2 domains. The SETD2 L1609P mutation is located in the SET domain within the SETD2 catalytic core (composed of the AWS, SET, and post-SET domains). Lower left panel : Structural representation of the SETD2 active site (PDB entry: 5JJY ) with a zoomed-in view of the substrate (H3K36M peptide) and cofactor (SAH) binding sites. Lower right panel : Sequence alignment of residues 1603 to 1619 of the SET domain of human SETD2 with the equivalent sequences of human G9A, EZH2, NSD1, NSD2, SETD8, MLL1, MLL2, SETD8, ASH1 (sequence retrieved from the UniProt database). Conserved residues are highlighted in blue . The secondary structure of the SETD2 residues (deduced from PDB entry: 5JJY ) is shown above the alignment. The SETD2 residue L1609 and the equivalent residues in the other SET domain-containing enzymes are highlighted in orange . B , in vitro methylation of recombinant histone H3, core histones (purified from HEK293T SETD2-KO cells) or recombinant nucleosomes. SETD2-dependent H3K36me3 methylation was detected using an anti-H3K36me3 antibody. Ponceau Red staining of histones is shown. The purified catalytic core of SETD2 WT and SETD2 L1609P mutant used in the assays were detected using an anti-6xHis-tag antibody. C , SETD2 mono-methylation, dimethylation, or trimethylation activities were determined by UFLC assays using H3K36 fluorescent peptides as previously described ( , ). Bar graphs and error bars represent the mean and SD of three independent experiments. D , automethylation of SETD2 and methylation of α-tubulin detected by autoradiography using 3 H-SAM. Coomassie Blue staining was used as loading control. E , determination of the intrinsic protein stability of SETD2 WT or SETD2 L1609P by thermal shift assay (TSA). Left panel : T m values were determined by the minimum of the first derivative of the fluorescence emission as a function of temperature (dFluo/dT). Right panel : Bar graphs and error bars represent the mean and SD of nine experiments. SETD2, SET-domain containing protein 2; UFLC, ultrafast liquid chromatography.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Activity Assay, In Vitro, Binding Assay, Sequencing, Residue, Methylation, Recombinant, Purification, Staining, Autoradiography, Control, Thermal Shift Assay, Fluorescence, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation results in low levels of the H3K36me3 mark and in low expression of SETD2 in CRISPR/Cas9-engineered HEK293T cells and in transfected HEK293T-SETD2 KO cells . A , endogenous H3K36me3 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : the H3K36me3 mark was detected by immunofluorescence using an anti-H3K36me3 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with 10 μm scale bars. Right panel : Histones from CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant were extracted and H3K36me3 levels were determined by Western blotting using a an anti-H3K36me3 antibody. Ponceau Red staining of extracted histones is shown. B , endogenous SETD2 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : Cells were fixed and SETD2 was detected using an anti-SETD2 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with scale bars of 10 μm. Right panel : SETD2 was detected in cell extracts by Western blot using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. C , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 L1609P were transfected with GFP-SETD2 WT or GFP-SETD2 L1609P plasmids. Nontransfected CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were used as controls. Ectopic GFP-SETD2 expression and H3K36me3 mark levels were detected by Western blot using anti-GFP or anti-H3K36me3 antibodies, respectively. Ponceau Red staining of cellular histones or extracts on membranes are shown. D , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were treated with MG132 or DMSO. Endogenous SETD2 WT and SETD2 L1609P expression levels were detected by Western blotting using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Expressing, CRISPR, Transfection, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Overall structure of the ternary complex of SETD2 L1609P mutant bound to H3K36M peptide and SAM cofactor . A , left panel : cartoon representation of SETD2 WT (PDB: 5JJY ) ( cyan ) bound to H3K36M peptide ( orange ) and the SAH cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 L1609 and H3M36 residues are represented by yellow and orange sticks , respectively. The close-up view shows the region around residue L1609 with the H3K36M peptide (residues 29–42, orange ) and the SAH cofactor ( black sticks ). Zinc atoms are shown in gray . Right panel : cartoon representation of the SETD2 L1609P mutant (PDB: 8RZU ) ( salmon ) bound to the H3K36M peptide ( green ) and the SAM cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 P1609 and H3M36 residues are shown as yellow and green sticks , respectively. The close-up view shows the region around the residue P1609 with the H3K36M peptide (residues 29–39, green ) and the SAM cofactor ( black sticks ). B , left panel : cartoon representation of the characteristic triangular shape of the SET domain formed by 3 β-sheets (β1-β2; β3-β8-β7; β4-β6-β5 strands) of SETD2 WT in complex with the H3K36M peptide (residues 29–42 in orange) (PDB: 5JJY ). The β-sheet composed of β4-β6-β5 strands is boxed and the SETD2 L1609 residue is shown in yellow . Right panel : cartoon representation of the triangular β-sheet structure of the SET domain of the SETD2 L1609P mutant ( salmon ) in complex with the H3K36M peptide (residues 29–39, green ) (PDB: 8RZU ). The β5-strand in SETD2 WT adopts a loop conformation in the structure of the SETD2 L1609P mutant ( boxed ). The P1609 residue in mutant SETD2 is shown in yellow . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Effects of the SETD2 L1609P mutation on the conformations of neighboring residues of SETD2 and the H3K36M peptide. A , the left panel shows a cartoon overlay of the β5-β6 hairpin of SETD2 WT (PDB: 5JJY ) ( cyan ) and SETD2 L1609P mutant ( salmon ) structures. The H3K36M peptide is shown in orange and green for SETD2 WT and SETD2 L1609P, respectively. The side chains of residues L1609 and P1609 residues are shown as sticks ( yellow CPK). The middle panel shows a close-up view of the hairpin residues (1609–1613) of SETD2 WT ( cyan ) and SETD2 L1609P ( salmon ). The side chains are shown in CPK sticks . The right panel shows the β5-β6 hairpin residues of SETD2 WT ( top ) and SETD2 L1609P ( bottom ) in sticks . Dashes represent the distance between Cα of residues K1610 and E1613 residues. B , conformational remodeling of residues K1610 and K1639 of SETD2 and residue K37 of H3 induced by the L1609P mutation. Left panel shows residues SETD2 L1609 ( yellow ), K1610 (cyan), K1639 ( cyan ), and H3K37 ( orange ) in spheres and sticks in the SETD2 WT structure (PDB: 5JJY ). Middle panel shows residues SETD2 P1609 ( yellow ), K1610 ( salmon ), K1639 ( salmon ), and H3K37 ( green ) in spheres and sticks in the SETD2 L1609P structure. The right panel shows residues P1609 ( yellow ) and K1610 ( salmon ) from the SETD2 L1609P structure and residues K1639 ( cyan ) and H3K37 ( orange ) from the SETD2 WT structure. Steric clashes between side chains are shown in boxes . The orientations are the same in all three panels and were obtained by superimposing the SETD2 WT and L1609P main chains. C , surface representation of the SETD2 substrate-binding region. H3K36M peptides are shown as sticks. The left panel shows the SETD2 WT structure (PDB: 5JJY ) in light cyan . The SETD2 L1609 residue is shown in yellow . The SETD2 K1610 and K1639 residues are shown in blue . H3K36M peptide residues diffracting in both WT and L1609P structures (residues A29–H39) are shown in green . H3K36M peptide residues observed only in the SETD2 WT structure (residues R40-R42) are shown in transparent orange . The right panel shows the SETD2 L1609P structure in light pink . The SETD2 P1609 residue is shown in yellow . The K1610 and K1639 residues are shown in purple . H3K36M peptide residues observed in the SETD2 L1609P structure (A29–H39) are shown in green . SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Details of H3K36M peptide recognition by SETD2 L1609P mutant . A , the left panel shows a clipped surface representation of the SETD2 WT-H3K36M peptide complex (PDB: 5JJY ). Peptide residues (residues A29–R42) are represented by sticks . The right panel shows a clipped surface representation of the SETD2 L1609P-H3K36M peptide complex. Peptide residues (A29–H39) are represented by sticks . The structures of the SETD2-H3K36M peptide complexes are shown in the same orientation after superimposition of the main chains. B , upper panel : Structural alignment of H3K36M peptides (residues A29–H39) in SETD2 WT (PDB: 5JJY ) ( orange ) and SETD2 L1609P ( green ) structures. Lower panel : Differences between SETD2-H3K36M peptide interactions in SETD2 WT and SETD2 L1609P complexes. Residue interactions across the binding interface of SETD2 WT or SETD2 L1609P mutant with H3K36M peptide were determined using LIGPLOT . Residues are represented by sticks . Residues involved in SETD2-H3K36M peptide interactions (nonbonded and hydrogen bonds) are represented by sticks and spheres . Dashes represent hydrogen bond. The lower left panel shows the SETD2 WT ( cyan )-H3M36 ( orange ) interacting residues that are specific for the SETD2 WT complex and not present in the SETD2 L1609P complex. These interactions are listed in a table ( bottom left ). The lower right panel shows SETD2 L1609P ( salmon )-H3K36M ( green ) peptide interacting residues that are specific for the SETD2 L1609P complex and not present in the SETD2 WT complex. These interactions are listed in a table ( bottom right ). SETD2, SET-domain containing protein 2.

Article Snippet: Five micromolars of recombinant SETD2 (WT, L1609P, or T1663M) were incubated ON at room temperature with 1 μg of recombinant human histone H3.3 (NEB) or 1 μg of recombinant human nucleosomes (16–0009, EpiCypher) or 2 μg core histones purified from HEK293T SETD2 KO cells , 100 μM SAM, and 1 mM DTT.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Summary of protein expression losses in ccRCC tumors

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Expressing

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: ( A ) How a phylogenetic tree was constructed. A: ARID1A loss; M: SMARCA2 loss; P: PBRM1 loss, G: SMARCA4 loss; S: SETD2 loss. ( B ) Truncal losses of the markers at each stage, either alone or in combination, were presented. ( C ) Fisher's exact tests were performed to calculate the p values of the associations between the protein marker losses and stages.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Construct, Marker

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: The survival curves were calculated based on SETD2 staining: positive (1) and negative (0). Associated log-rank p value was indicated. n: number of cases.

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Staining

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with overall survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: Oncotarget

Article Title: Intratumoral heterogeneity analysis reveals hidden associations between protein expression losses and patient survival in clear cell renal cell carcinoma

doi: 10.18632/oncotarget.16965

Figure Lengend Snippet: Univariate and multivariable analyses of indicated biomarker losses and their associations with recurrence-free survival

Article Snippet: The antibodies used for IHC are: PBRM1 (Bethyl labs, Cat# A301-591A), ARID1A (Sigma-Aldrich, Cat# HPA005456), SMARCA2 (Sigma-Aldrich, Cat# HPA029981), SMARCA4 (Abcam, Cat# ab110641), SETD2 (ProSci, Cat# 30-305).

Techniques: Biomarker Discovery

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: Primary antibodies used for indirect immunofluorescence

Article Snippet: SETD2 , Rabbit , 1:500 , Knockdown validated , Cell Signaling Technology, 89680S.

Techniques: Biomarker Discovery, Knock-Out, Knockdown, Mutagenesis, Membrane

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: SETD2 and H3K36me3 peak in early growing oocytes before declining during late oocyte growth. ( A , C ) Representative IF images showing SETD2 (A) and H3K36me3 (C) (red, grey single channel) in primordial, primary, secondary, early antral and antral follicle oocytes from wildtype adult mouse ovaries. The oocyte nucleus is defined by Lamin B1 (green, dashed lines), and DNA is shown by DAPI (blue). White boxes indicate regions shown at higher power in images on the right. Yellow arrowheads denote primordial follicle oocyte nuclei. Images are representative of multiple planes in both ovaries from three biological replicates. Scale bars represent 50 μm (left), and 10 μm (middle, right). ( B , D ) Quantification of SETD2 (B) and H3K36me3 (D) within oocyte nuclei of primordial, primary, secondary, early antral and antral follicles from wildtype adult mouse ovaries. Values represent average nuclear staining intensity with average cytoplasmic fluorescence removed to correct for non-specific background staining. Error bars represent mean ± SD. ( B ) ns: not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA plus Tukey’s multiple comparisons test. N = 94 primordial follicle oocytes, N = 56 primary follicle oocytes, N = 32 secondary follicle oocytes, N = 17 early antral follicle oocytes, and N = 13 antral follicle oocytes, in total from three biological replicates. ( D ) ns: not significant, * P < 0.05, *** P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons test. N = 82 primordial follicle oocytes, N = 47 primary follicle oocytes, N = 21 secondary follicle oocytes, N = 11 early antral follicle oocytes, and N = 6 antral follicle oocytes, in total from three biological replicates

Article Snippet: SETD2 , Rabbit , 1:500 , Knockdown validated , Cell Signaling Technology, 89680S.

Techniques: Staining, Fluorescence

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: Loss of EED significantly increased SETD2 in primary, secondary, and early antral follicle oocytes. ( A ) Representative IF images showing SETD2 (red, grey single channel) in primary, secondary, early antral and antral follicle oocytes from Eed -wt (left), Eed -het (middle), and Eed -hom (right) adult mouse ovaries. The oocyte nucleus is defined by Lamin B1 (green, dashed lines), and DNA is shown by DAPI (blue). Images are representative of multiple planes in both ovaries from three biological replicates for each genotype. Scale bars represent 50 μm. ( B-E ) Quantification of SETD2 within oocyte nuclei of primary ( B ), secondary ( C ), early antral ( D ), and antral ( E ) follicles from Eed -wt, Eed -het, and Eed -hom adult mouse ovaries. Data is from three biological replicates for each genotype. Values represent average nuclear staining intensity with average cytoplasmic fluorescence removed to correct for non-specific background staining. Average intensity for Eed -het and Eed -hom samples are shown relative to Eed -wt set to 1.0. Error bars represent mean ± SD. ( B-C ) N = 56 Eed -wt, N = 44 Eed -het, N = 39 Eed -hom primary follicle oocytes. N = 32 Eed -wt, N = 17 Eed -het, N = 31 Eed -hom secondary follicle oocytes. ns: not significant, ** P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons test. ( D-E ) N = 17 Eed -wt, N = 11 Eed -het, N = 6 Eed -hom early antral follicle oocytes. N = 13 Eed -wt, N = 10 Eed -het, N = 18 Eed -hom antral follicle oocytes. ns: not significant, * P < 0.05, one-way ANOVA plus Tukey’s multiple comparisons test

Article Snippet: SETD2 , Rabbit , 1:500 , Knockdown validated , Cell Signaling Technology, 89680S.

Techniques: Staining, Fluorescence

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A gene expression based predictor for myeloma patients at high risk of developing bone disease on bisphosphonate treatment

doi: 10.1158/1078-0432.CCR-11-0994

Figure Lengend Snippet: Fourteen genes (15 probesets) upregulated in patients who developed on-treatment SREs (ranked by SAM score).

Article Snippet: Some of these were interferon-induced genes, and the others mainly involved in cell signalling and mitosis. table ft1 table-wrap mode="anchored" t5 caption a7 Affymetrix Probeset Gene Symbol SAM Score FC Pathway/Function Cytoband 205269_at LCP2 3.93 2.03 Cell signalling 5q33.1-qter 209799_at PRKAA1 3.84 1.47 Cell signalling 5p12 231771_at GJB6 3.8 1.53 Cell signalling, apoptosis 13q11-q12.1|13q12 226757_at IFIT2 3.69 1.72 Interferon-induced protein 10q23-q25 205220_at NIACR2 3.68 1.55 Cell signalling 12q24.31 219947_at CLEC4A 3.54 1.7 Cell signalling, adhesion 12p13 229450_at IFIT3 3.53 2.58 Interferon-induced protein 10q24 209137_s_at USP10 3.48 1.33 DNA repair 16q24.1 222235_s_at CSGALNACT 2 /// RP1- 19N1.1 3.41 1.56 Not known 10q11.21 /// Xq22.3 227609_at EPSTI1 3.39 1.61 Interferon response gene 13q13.3 204162_at NDC80 3.35 1.85 Cell mitosis 18p11.32 209408_at KIF2C 3.31 1.3 Cell mitosis 1p34.1 238903_at UBXN2B 3.31 1.3 Not known 8q12.1 225519_at PPP4R2 3.3 1.39 Cell mitosis 3p13 225984_at PRKAA1 3.29 1.33 Cell signalling 5p12 Open in a separate window Fourteen genes (15 probesets) upregulated in patients who developed on-treatment SREs (ranked by SAM score).

Techniques:

Journal: Nucleic Acids Research

Article Title: Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing

doi: 10.1093/nar/gkaa556

Figure Lengend Snippet: ( A ) Setd2 mRNA levels assessed by RT-qPCR 48 h post transfection. All data are normalised to Ct values for Actb . Data are given as mean 2 −ΔΔCt values ± SEM of three independent experiments. *** P < 0.001 compared with Scrambled group by unpaired t -test. Scr, scrambled control; KD, knockdown. ( B ) Western blot of whole cell lysate showing effective depletion of H3K36me3 upon knockdown of Setd2 . Total histone H3 levels are unaffected. ACTB was used as a loading control. Untr, untreated cells; Lipo, transfection vehicle only; Scr, scrambled control; KD, knockdown. ( C ) RNA-seq heatmap of significant differentially expressed transcripts 48 h post transfection. Values are given as row-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Scr, scrambled control; KD, knockdown. ( D ) Top ten upregulated biological processes determined by GO analysis (PANTHER). See for a complete list of GO ID terms. ( E ) Top ten downregulated biological processes determined by GO analysis (PANTHER). See for a complete list of GO ID terms.

Article Snippet: Setd2 -specific or Scrambled siRNAs (OriGene, SR423523) were diluted in 125 μl Opti-MEM to a final concentration of 10 nM.

Techniques: Quantitative RT-PCR, Transfection, Control, Knockdown, Western Blot, RNA Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation decreases methyltransferase activity and intrinsic protein stability of SETD2 catalytic core in vitro . A , upper panel : schematic representation of the SETD2 domains. The SETD2 L1609P mutation is located in the SET domain within the SETD2 catalytic core (composed of the AWS, SET, and post-SET domains). Lower left panel : Structural representation of the SETD2 active site (PDB entry: 5JJY ) with a zoomed-in view of the substrate (H3K36M peptide) and cofactor (SAH) binding sites. Lower right panel : Sequence alignment of residues 1603 to 1619 of the SET domain of human SETD2 with the equivalent sequences of human G9A, EZH2, NSD1, NSD2, SETD8, MLL1, MLL2, SETD8, ASH1 (sequence retrieved from the UniProt database). Conserved residues are highlighted in blue . The secondary structure of the SETD2 residues (deduced from PDB entry: 5JJY ) is shown above the alignment. The SETD2 residue L1609 and the equivalent residues in the other SET domain-containing enzymes are highlighted in orange . B , in vitro methylation of recombinant histone H3, core histones (purified from HEK293T SETD2-KO cells) or recombinant nucleosomes. SETD2-dependent H3K36me3 methylation was detected using an anti-H3K36me3 antibody. Ponceau Red staining of histones is shown. The purified catalytic core of SETD2 WT and SETD2 L1609P mutant used in the assays were detected using an anti-6xHis-tag antibody. C , SETD2 mono-methylation, dimethylation, or trimethylation activities were determined by UFLC assays using H3K36 fluorescent peptides as previously described ( , ). Bar graphs and error bars represent the mean and SD of three independent experiments. D , automethylation of SETD2 and methylation of α-tubulin detected by autoradiography using 3 H-SAM. Coomassie Blue staining was used as loading control. E , determination of the intrinsic protein stability of SETD2 WT or SETD2 L1609P by thermal shift assay (TSA). Left panel : T m values were determined by the minimum of the first derivative of the fluorescence emission as a function of temperature (dFluo/dT). Right panel : Bar graphs and error bars represent the mean and SD of nine experiments. SETD2, SET-domain containing protein 2; UFLC, ultrafast liquid chromatography.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Activity Assay, In Vitro, Binding Assay, Sequencing, Residue, Methylation, Recombinant, Purification, Staining, Autoradiography, Control, Thermal Shift Assay, Fluorescence, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: The L1609P mutation results in low levels of the H3K36me3 mark and in low expression of SETD2 in CRISPR/Cas9-engineered HEK293T cells and in transfected HEK293T-SETD2 KO cells . A , endogenous H3K36me3 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : the H3K36me3 mark was detected by immunofluorescence using an anti-H3K36me3 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with 10 μm scale bars. Right panel : Histones from CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant were extracted and H3K36me3 levels were determined by Western blotting using a an anti-H3K36me3 antibody. Ponceau Red staining of extracted histones is shown. B , endogenous SETD2 levels in CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or L1609P mutant. Left panel : Cells were fixed and SETD2 was detected using an anti-SETD2 antibody. DAPI staining was used for nuclei localization. Optical sections are shown with scale bars of 10 μm. Right panel : SETD2 was detected in cell extracts by Western blot using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. C , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 L1609P were transfected with GFP-SETD2 WT or GFP-SETD2 L1609P plasmids. Nontransfected CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were used as controls. Ectopic GFP-SETD2 expression and H3K36me3 mark levels were detected by Western blot using anti-GFP or anti-H3K36me3 antibodies, respectively. Ponceau Red staining of cellular histones or extracts on membranes are shown. D , CRISPR/Cas9-engineered HEK293T cells expressing SETD2 WT or SETD2 L1609P were treated with MG132 or DMSO. Endogenous SETD2 WT and SETD2 L1609P expression levels were detected by Western blotting using an anti-SETD2 antibody. Ponceau Red staining of the cell extracts is shown. SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Expressing, CRISPR, Transfection, Immunofluorescence, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Overall structure of the ternary complex of SETD2 L1609P mutant bound to H3K36M peptide and SAM cofactor . A , left panel : cartoon representation of SETD2 WT (PDB: 5JJY ) ( cyan ) bound to H3K36M peptide ( orange ) and the SAH cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 L1609 and H3M36 residues are represented by yellow and orange sticks , respectively. The close-up view shows the region around residue L1609 with the H3K36M peptide (residues 29–42, orange ) and the SAH cofactor ( black sticks ). Zinc atoms are shown in gray . Right panel : cartoon representation of the SETD2 L1609P mutant (PDB: 8RZU ) ( salmon ) bound to the H3K36M peptide ( green ) and the SAM cofactor ( gray sticks ). The protein surface is shown as transparent. The side chains of the SETD2 P1609 and H3M36 residues are shown as yellow and green sticks , respectively. The close-up view shows the region around the residue P1609 with the H3K36M peptide (residues 29–39, green ) and the SAM cofactor ( black sticks ). B , left panel : cartoon representation of the characteristic triangular shape of the SET domain formed by 3 β-sheets (β1-β2; β3-β8-β7; β4-β6-β5 strands) of SETD2 WT in complex with the H3K36M peptide (residues 29–42 in orange) (PDB: 5JJY ). The β-sheet composed of β4-β6-β5 strands is boxed and the SETD2 L1609 residue is shown in yellow . Right panel : cartoon representation of the triangular β-sheet structure of the SET domain of the SETD2 L1609P mutant ( salmon ) in complex with the H3K36M peptide (residues 29–39, green ) (PDB: 8RZU ). The β5-strand in SETD2 WT adopts a loop conformation in the structure of the SETD2 L1609P mutant ( boxed ). The P1609 residue in mutant SETD2 is shown in yellow . SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Effects of the SETD2 L1609P mutation on the conformations of neighboring residues of SETD2 and the H3K36M peptide. A , the left panel shows a cartoon overlay of the β5-β6 hairpin of SETD2 WT (PDB: 5JJY ) ( cyan ) and SETD2 L1609P mutant ( salmon ) structures. The H3K36M peptide is shown in orange and green for SETD2 WT and SETD2 L1609P, respectively. The side chains of residues L1609 and P1609 residues are shown as sticks ( yellow CPK). The middle panel shows a close-up view of the hairpin residues (1609–1613) of SETD2 WT ( cyan ) and SETD2 L1609P ( salmon ). The side chains are shown in CPK sticks . The right panel shows the β5-β6 hairpin residues of SETD2 WT ( top ) and SETD2 L1609P ( bottom ) in sticks . Dashes represent the distance between Cα of residues K1610 and E1613 residues. B , conformational remodeling of residues K1610 and K1639 of SETD2 and residue K37 of H3 induced by the L1609P mutation. Left panel shows residues SETD2 L1609 ( yellow ), K1610 (cyan), K1639 ( cyan ), and H3K37 ( orange ) in spheres and sticks in the SETD2 WT structure (PDB: 5JJY ). Middle panel shows residues SETD2 P1609 ( yellow ), K1610 ( salmon ), K1639 ( salmon ), and H3K37 ( green ) in spheres and sticks in the SETD2 L1609P structure. The right panel shows residues P1609 ( yellow ) and K1610 ( salmon ) from the SETD2 L1609P structure and residues K1639 ( cyan ) and H3K37 ( orange ) from the SETD2 WT structure. Steric clashes between side chains are shown in boxes . The orientations are the same in all three panels and were obtained by superimposing the SETD2 WT and L1609P main chains. C , surface representation of the SETD2 substrate-binding region. H3K36M peptides are shown as sticks. The left panel shows the SETD2 WT structure (PDB: 5JJY ) in light cyan . The SETD2 L1609 residue is shown in yellow . The SETD2 K1610 and K1639 residues are shown in blue . H3K36M peptide residues diffracting in both WT and L1609P structures (residues A29–H39) are shown in green . H3K36M peptide residues observed only in the SETD2 WT structure (residues R40-R42) are shown in transparent orange . The right panel shows the SETD2 L1609P structure in light pink . The SETD2 P1609 residue is shown in yellow . The K1610 and K1639 residues are shown in purple . H3K36M peptide residues observed in the SETD2 L1609P structure (A29–H39) are shown in green . SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The SETD2 L1609P mutation found in leukemia disrupts methyltransferase activity and reduces histone H3K36 trimethylation

doi: 10.1016/j.jbc.2026.111259

Figure Lengend Snippet: Details of H3K36M peptide recognition by SETD2 L1609P mutant . A , the left panel shows a clipped surface representation of the SETD2 WT-H3K36M peptide complex (PDB: 5JJY ). Peptide residues (residues A29–R42) are represented by sticks . The right panel shows a clipped surface representation of the SETD2 L1609P-H3K36M peptide complex. Peptide residues (A29–H39) are represented by sticks . The structures of the SETD2-H3K36M peptide complexes are shown in the same orientation after superimposition of the main chains. B , upper panel : Structural alignment of H3K36M peptides (residues A29–H39) in SETD2 WT (PDB: 5JJY ) ( orange ) and SETD2 L1609P ( green ) structures. Lower panel : Differences between SETD2-H3K36M peptide interactions in SETD2 WT and SETD2 L1609P complexes. Residue interactions across the binding interface of SETD2 WT or SETD2 L1609P mutant with H3K36M peptide were determined using LIGPLOT . Residues are represented by sticks . Residues involved in SETD2-H3K36M peptide interactions (nonbonded and hydrogen bonds) are represented by sticks and spheres . Dashes represent hydrogen bond. The lower left panel shows the SETD2 WT ( cyan )-H3M36 ( orange ) interacting residues that are specific for the SETD2 WT complex and not present in the SETD2 L1609P complex. These interactions are listed in a table ( bottom left ). The lower right panel shows SETD2 L1609P ( salmon )-H3K36M ( green ) peptide interacting residues that are specific for the SETD2 L1609P complex and not present in the SETD2 WT complex. These interactions are listed in a table ( bottom right ). SETD2, SET-domain containing protein 2.

Article Snippet: A pet28a-MHL plasmid containing the cDNA coding for the human SETD2 catalytic domain (Addgene #25348, residues 1433–1711) was used in order to produce 6xHis-tagged WT SETD2 in BL21 HI-control (DE3) E . coli .

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Cell reports

Article Title: Menin orchestrates macrophage reprogramming to maintain the pulmonary immune homeostasis.

doi: 10.1016/j.celrep.2024.115219

Figure Lengend Snippet: Figure 7. Interaction of menin/SETD2 contributes to LPS-induced activation of AM (A) Protein levels were detected in BALF from Men1f/f and Men1DM/DM mice. Mice were treated with saline or LPS (5 mg/kg) through intratracheal injection for 24 h (n = 6). (B) RT-qPCR was used to analyze the mRNA expression of Csf2, Il-6, Il-1b, Cxcl15, Tnf, and Men1 in AMs from BALF of Men1f/f and Men1DM/DM mice. Mice were treated with saline or LPS, relative to (A) (n = 6). (C) ChIP-qPCR was performed with indicated antibodies in BMDM cells from Men1f/f and Men1DM/DM mice. BMDM cells were treated with or without LPS (100 ng/ mL) for 48 h, and IgG served as the negative control (n = 3). (D) Representative H&E and IHC (F4/80) staining on paraffin sections from lung tissue of WT-C57BL/6J mice. Mice were treated with saline or LPS (5 mg/kg) through intratracheal injection for 24 h. Therapeutically, mice were pre-treated with EZM0414 or pep242 respectively (n = 6, scale bar, 100 mm). (E) BALF protein levels detection from above-mentioned lung injury models in (D). (F) IP was performed in AMs from BALF of WT-C57BL/6J mice with anti-menin antibody. AMs were pre-treated with pep242 (50 mg/mL) for 24 h, followed by treatment with or without LPS (100 ng/mL) for 24 h. Western blotting was used to detect the menin and SETD2. Depicted in (A)–(C), and (E) are mean and SD. Statistical significance was determined by ANOVA with Tukey’s multiple comparisons (A–C, E). Results in (F) are representative of three independent experi- ments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FITC anti-mouse CD45.2 Antibody Biolegend Cat# 109805; RRID: AB_313442 PE/Cyanine7 anti-mouse CD45.1 Antibody Biolegend Cat# 110729; RRID: AB_1134168 Bacterial and virus strains Lentivirus-expressing shMEN1-1 This paper N/A Lentivirus-expressing shMEN1-2 This paper N/A pET-28a vector This paper N/A Biological samples Mouse tissues This paper N/A Chemicals, peptides, and recombinant proteins Thioglycolate medium Fluka 70157 Tamoxifen Sigma T5648-5G Corn oil Sigma C8267 pGMLV-SV40T lentiviral Genemeditech GM-0220SV01 RIPA lysis buffer Beyotime P0013B NP-40 lysis buffer Beyotime P0013F ACK Lysis buffer Beyotime C3702 Pepstatin MCE HY-P0017 Aprotinin MCE HY-P0018 Cell staining buffer Biolegend 420201 MG132 Selleck S2619 Cycloheximide (CHX) Sigma C7698-1g Polyethyleneimine (PEI) Polyscences 23966-1g Polybrene Sigma H9268 DAPI VECTOR H-1200 Recombinant Murine M-CSF Peprotech 315–02 Recombinant human M-CSF Peprotech 300–25 Recombinant Mouse GM-CSF R&D system 415-ML-005 Recombinant Murine IL-4 Peprotech 214–14 Recombinant Murine IL-13 Peprotech 210–13 InVivoMab anti-mouse GM-CSF Bioxcell BE0259 LPS Sigma L6529 IFNg Peprotech 315–05 MI-3 Selleck S7619 Sinefungin J&K Scientific JK186839-1MG JIB-04 yuanye Bio Technology S81423-5MG EZM0414-TFA MCE HY-136328 PMA Sigma P1585-1mg Pep242 This paper N/A Menin231-248 This paper N/A Critical commercial assays ChIP Assay kit Millipore 17–295 IHC kit Maxim, China 9370, 9708 RT-PCR Vazyme R323 Mut Express II Fast Mutagenesis Kit Vazyme C214 His-tag Protein Purification Kit Beyotime P2226 Mouse GM-CSF ELISA Kit MULTI SCIENCES EK263/2-96 DAB staining kit Maxim, China 2031 Periodic Acid-Schiff Staining Kit Beyotime C0142S (Continued on next page) Cell Reports 44, 115219, January 28, 2025 15

Techniques: Activation Assay, Saline, Injection, Quantitative RT-PCR, Expressing, ChIP-qPCR, Negative Control, Staining, Western Blot