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: The EMBO Journal

Article Title: The H3K36me3 methyltransferase SETD2 contributes to PAF1C interactions with RNA Pol II and is required for neuronal differentiation

doi: 10.1038/s44318-026-00768-2

Figure Lengend Snippet: ( A ) Top: schematics depicting the neuronal differentiation protocol. Bottom: cell count assay using live-dead stain at the cellular aggregate stage day 4 (CAd4), neural progenitor cells (NPC), and terminal neurons stage day 14 (TN). Depicted are percentages of survival in WT, Setd2 -KO, Eed -KO, Dmnt -TKO, and QKO cells, calibrated to 100% survival in WT cells. Data obtained from three to five independent replicates is shown. Asterisks indicate P values calculated using a two-tailed t test. For CAd4: *=5E-07, **=0.013, and ***=0.011; for NPC: *=0.012, **=1E-06, ***=2E-05, and ****=4E-05; for TN: *=2E-08, **=1E-09, ***=2E-08, and ****=1E-08. ( B ) Microscopy images of WT and Setd2 -KO TNs at day d14 at ×100 magnification. Average percentages of survival after dissociation obtained from three independent experiments are indicated. ( C ) Same as in ( B ) but using TNs from Tet-inducible Setd2 knockdown cell lines that were treated with 1 μg/ml doxycycline (DOX) from d0 to d14, or from d10 to d14. Continuous knockdown of Setd2 results in neuronal cell death, while knockdown in mature neurons does not influence survival. Scale bars, 50 µm. .

Article Snippet: Membranes were blocked with 5% milk in TBS with 0.1% Tween20 and incubated with primary antibodies against SETD2 (1:1000, A3194, ABclonal, LOT 0071240201), H3K36me3 (1:5000, ab9050, Abcam, LOT GR3210075-1), H3K79me2 (1:2000, 04-835, Milipore, clone NL-59), H2BK120ub (1:2000, MM-0029-P, Medimabs, clone NRO3), anti-histone H1 (1:5000, AE04, Millipore, LOT 3087175), anti-NEUROG1 (1:1000, sc-100332, Santa Cruz Biotechnology, LOT F1119), anti-LAMIN B1 (1:1000, sc-374015, Santa Cruz Biotechnology, LOT J3019), anti-HA (1:1000, ab9110, Abcam), Streptavidin-HRP (1:20000, Pierce, clone 21130), overnight at 4 °C.

Techniques: Cell Characterization, Staining, Two Tailed Test, Microscopy, Knockdown

Journal: The EMBO Journal

Article Title: The H3K36me3 methyltransferase SETD2 contributes to PAF1C interactions with RNA Pol II and is required for neuronal differentiation

doi: 10.1038/s44318-026-00768-2

Figure Lengend Snippet: ( A ) MA plot of bulk PolyA-RNA-seq results depicting differences in gene expression between wild-type (WT) and Setd2 -KO NPCs ( P value < 0.05, LFC > I1I). ( B ) Motif Activity Response Analysis identifies transcription factor motifs preferentially bound by Rest and Sox2 in the promoters of genes downregulated in Setd2 -KO NPCs, and motifs coinciding with E2f1 and Myc/Max transcription factor binding in the promoters of upregulated genes. ( C ) UMAP projection of single-cell RNA seq data obtained from wild-type and Setd2 -KO ESCs and NPCs. Shown are annotations of different cell states obtained from grouping unsupervised clusters shown in Appendix Fig. . ( D ) Same UMAP projection as in ( C ) but showing the individual cell lines and cell types. ( E ) Expression of Myt1 and Uncx is predominantly observed in differentiating wild-type neuronal cells.

Article Snippet: Membranes were blocked with 5% milk in TBS with 0.1% Tween20 and incubated with primary antibodies against SETD2 (1:1000, A3194, ABclonal, LOT 0071240201), H3K36me3 (1:5000, ab9050, Abcam, LOT GR3210075-1), H3K79me2 (1:2000, 04-835, Milipore, clone NL-59), H2BK120ub (1:2000, MM-0029-P, Medimabs, clone NRO3), anti-histone H1 (1:5000, AE04, Millipore, LOT 3087175), anti-NEUROG1 (1:1000, sc-100332, Santa Cruz Biotechnology, LOT F1119), anti-LAMIN B1 (1:1000, sc-374015, Santa Cruz Biotechnology, LOT J3019), anti-HA (1:1000, ab9110, Abcam), Streptavidin-HRP (1:20000, Pierce, clone 21130), overnight at 4 °C.

Techniques: RNA Sequencing, Gene Expression, Activity Assay, Binding Assay, Single Cell, Expressing

Journal: The EMBO Journal

Article Title: The H3K36me3 methyltransferase SETD2 contributes to PAF1C interactions with RNA Pol II and is required for neuronal differentiation

doi: 10.1038/s44318-026-00768-2

Figure Lengend Snippet: ( A ) Schematic overview of mESC line generation harboring a Tet-inducible Neurogenin 2-Neurogenin 1 (iNgn) master transcription factor cassette in a Setd2 -KO background using recombination-mediated cassette exchange. ( B ) Microscopy images of in vitro - derived neurons (d14) show successful differentiation of neuronal cells derived from wild-type ESCs and partial rescue of cell death in Setd2 -deficient cells by overexpression of an inducible Neurogenin 2-Neurogenin 1 fusion factor (i Ngn ). Similar results were obtained from two independent replicates. Cells were treated with 1 μg/ml doxycycline ( + DOX) during the entire in vitro differentiation, or with DMSO (no DOX). Mean neuronal survival rate (in percent) from three independent replicates is shown. ( C ) RT-qPCR of various lineage markers in WT and Setd2 -KO NPCs, the latter expressing Neurogenin 1 and Neurogenin 2 upon 1 µg/ml doxycycline treatment ( n = 3). Target genes: Oct4 ( Pou5f1 ), Nanog - embryonic stem cell markers; Pax3 , Pax6 , Ngn1 , Crabp1 , Msx3 , Foxd3 - neuronal marker; Fgf4 - growth factor, Sox17 - parietal endoderm marker. Gene expression was calibrated to Hprt - housekeeping gene expression. Bars and whiskers indicate means and SD. .

Article Snippet: Membranes were blocked with 5% milk in TBS with 0.1% Tween20 and incubated with primary antibodies against SETD2 (1:1000, A3194, ABclonal, LOT 0071240201), H3K36me3 (1:5000, ab9050, Abcam, LOT GR3210075-1), H3K79me2 (1:2000, 04-835, Milipore, clone NL-59), H2BK120ub (1:2000, MM-0029-P, Medimabs, clone NRO3), anti-histone H1 (1:5000, AE04, Millipore, LOT 3087175), anti-NEUROG1 (1:1000, sc-100332, Santa Cruz Biotechnology, LOT F1119), anti-LAMIN B1 (1:1000, sc-374015, Santa Cruz Biotechnology, LOT J3019), anti-HA (1:1000, ab9110, Abcam), Streptavidin-HRP (1:20000, Pierce, clone 21130), overnight at 4 °C.

Techniques: Microscopy, In Vitro, Derivative Assay, Over Expression, Quantitative RT-PCR, Expressing, Marker, Gene Expression

Journal: The EMBO Journal

Article Title: The H3K36me3 methyltransferase SETD2 contributes to PAF1C interactions with RNA Pol II and is required for neuronal differentiation

doi: 10.1038/s44318-026-00768-2

Figure Lengend Snippet: ( A ) Representative genome browser view exemplifying differences in ChIP-seq signal of various chromatin marks and ATAC-seq signal between wild-type and Setd2 -KO NPCs. Shown are read counts per 100 bp for ChIP-seq samples. Arrow exemplifies a downregulated neuronal gene Ncan . ( B ) Scatterplot showing a minor correlation between the measured H4-panacetylation changes in Setd2 -KO over WT NPCs and H3K36me3 levels at gene bodies measured in WT cells. Pearson’s correlation coefficient is shown. ( C ) Scatterplot showing changes in gene expression measured in Setd2 -KO over WT NPCs and their relation to H3K36me3 levels at gene bodies measured in WT cells. Pearson’s correlation coefficient is shown. ( D ) Metaprofile plots of RNA Pol II ChIP-seq data obtained from wild-type and Setd2 -KO NPCs at genes up- and downregulated in the absence of SETD2. Downregulated genes show reduced Pol II signals in the absence of SETD2. ( E ) Cell survival assay using live-dead stain at CAd4, NPC, and TN stage (d14). Depicted are percentages of survival for WT, Setd2 -KO, and SET domain catalytic mutant (R1599H) cells. Bars and whiskers indicate means and SD. Asterisks indicate P values calculated using a two-tailed t test: *=4E-011 and **=2E-06. .

Article Snippet: Membranes were blocked with 5% milk in TBS with 0.1% Tween20 and incubated with primary antibodies against SETD2 (1:1000, A3194, ABclonal, LOT 0071240201), H3K36me3 (1:5000, ab9050, Abcam, LOT GR3210075-1), H3K79me2 (1:2000, 04-835, Milipore, clone NL-59), H2BK120ub (1:2000, MM-0029-P, Medimabs, clone NRO3), anti-histone H1 (1:5000, AE04, Millipore, LOT 3087175), anti-NEUROG1 (1:1000, sc-100332, Santa Cruz Biotechnology, LOT F1119), anti-LAMIN B1 (1:1000, sc-374015, Santa Cruz Biotechnology, LOT J3019), anti-HA (1:1000, ab9110, Abcam), Streptavidin-HRP (1:20000, Pierce, clone 21130), overnight at 4 °C.

Techniques: ChIP-sequencing, Gene Expression, Clonogenic Cell Survival Assay, Staining, Mutagenesis, Two Tailed Test

Journal: The EMBO Journal

Article Title: The H3K36me3 methyltransferase SETD2 contributes to PAF1C interactions with RNA Pol II and is required for neuronal differentiation

doi: 10.1038/s44318-026-00768-2

Figure Lengend Snippet: ( A ) Schematic overview of the ChromID setup using SETD2-SRI domain fused to TurboID to detect protein-protein interactions on the elongating RNA Pol II in wild-type and Setd2 -KO NPCs using mass spectrometry. ( B ) Heatmap indicating the combined significantly enriched proteins in the wild-type and Setd2 -KO NPCs sample over a background control expressing NLS-TurboID. Results obtained from four independent replicates. Colors indicate log2-fold change in LFQ intensity from SETD2-SRI over NLS-TurboID in the respective experiments. SETD2 peptide counts display combined tryptic peptides from endogenous SETD2 and SRI-TurboID. ( C ) Venn diagram indicating the overlap between proteins significantly enriched by SRI-TurboID over nTurbo in Setd2-KO and wild-type cells. (FDR-corrected two-tailed t test: FDR = 0.01, s0 = 0.1, log 2 FC > 0, n = 4 independent replicates). ( D ) STRING analysis of proteins that show reduced RNA Pol II interactions in Setd2 -KO NPCs when compared to wild-type NPCs identifies factors involved in RNA Pol II elongation. ( E ) Bar plot representing the top ten Molecular Function GO terms summarizing the proteins depleted from RNA Pol II in Setd2 -KO NPCs. Data was generated using the Enrichr database. .

Article Snippet: Membranes were blocked with 5% milk in TBS with 0.1% Tween20 and incubated with primary antibodies against SETD2 (1:1000, A3194, ABclonal, LOT 0071240201), H3K36me3 (1:5000, ab9050, Abcam, LOT GR3210075-1), H3K79me2 (1:2000, 04-835, Milipore, clone NL-59), H2BK120ub (1:2000, MM-0029-P, Medimabs, clone NRO3), anti-histone H1 (1:5000, AE04, Millipore, LOT 3087175), anti-NEUROG1 (1:1000, sc-100332, Santa Cruz Biotechnology, LOT F1119), anti-LAMIN B1 (1:1000, sc-374015, Santa Cruz Biotechnology, LOT J3019), anti-HA (1:1000, ab9110, Abcam), Streptavidin-HRP (1:20000, Pierce, clone 21130), overnight at 4 °C.

Techniques: Protein-Protein interactions, Mass Spectrometry, Control, Expressing, Two Tailed Test, Generated

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