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: Leukemia

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

doi: 10.1038/leu.2017.183

Figure Lengend Snippet: SETD2 loss of function mutations in the index MCL case (MCL1). ( a ) Sanger sequencing chromatograms with the frameshift (top) and nonsense (bottom) mutations identified by whole-exome sequencing. ( b ) Localization of the mutations with respect to the key functional domains of the SETD2 protein. The SRI domain is necessary for histone H3 lysine 36 trimethylation (H3K36Me3) and mediates SETD2 interaction with the phosphorylated C-terminal domain of the RNA polymerase II large subunit (RNA pol II) and with the heterogeneous nuclear ribonucleoprotein-L (HnRNP), thus coupling H3K36Me3 with transcription elongation and splicing. ( c ) from top to bottom: western blotting (WB) showing the truncated SETD2 (tSETD2) protein as compared to full-length SETD2 detectable in a pool of proteins from mononuclear cells of healthy donors; co-immunoprecipitation experiments performed by using: an anti-RNA pol II, an anti-hnRNP and an anti-histone H3, respectively, to isolate the proteins of interest and then an anti-SETD2 as primary antibody to label the PVDF membrane on which the immunoprecipitates were transferred; WB for H3K36Me3. Histone H3 and actin were used as loading controls.

Article Snippet: Assays were performed in triplicate on the ABI 7900HT system (Thermo Fisher Scientific) using pre-designed TaqMan Gene Expression Assays (Thermo Fisher Scientific) for SETD2 (Hs01014784_m1) and TBP (Hs00427621_m1) as control gene.

Techniques: Sequencing, Functional Assay, Western Blot, Immunoprecipitation, Membrane

Journal: Leukemia

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

doi: 10.1038/leu.2017.183

Figure Lengend Snippet: SETD2 protein and H3K36Me3 deficiency in SM. ( a ) representative western blot results for SETD2 protein and H3K36Me3 levels in SM patients as compared to a pool of healthy donors (HDs). One of three independent experiments is shown. ( b and c ) Box and whiskers plots of SETD2 and H3K36Me3 level estimates obtained by densitometric analysis of western blots. Median, interquartile range, minimum, maximum and outliers are indicated. SETD2 and H3K36Me3 signal intensities in single blots obtained from three individual experiments were normalized to those of beta-actin and H3 histone, respectively, and averaged. Normalized SETD2 and H3K36Me3 levels calculated in SM patients were then expressed in comparison to normalized SETD2 and H3K36Me3 levels detected in a pool of HDs, conventionally set to 1. The asterisks indicate that MCL and ASM had significantly lower levels of SETD2 protein ( P <0.001 and P =0.002, respectively) and H3K36Me3 ( P <0.001 and P =0.004, respectively) as compared to ISM.

Article Snippet: Assays were performed in triplicate on the ABI 7900HT system (Thermo Fisher Scientific) using pre-designed TaqMan Gene Expression Assays (Thermo Fisher Scientific) for SETD2 (Hs01014784_m1) and TBP (Hs00427621_m1) as control gene.

Techniques: Western Blot, Comparison

Journal: Leukemia

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

doi: 10.1038/leu.2017.183

Figure Lengend Snippet: SETD2 protein and H3K36Me3 levels in HMC-1.1 and -1.2 cell lines before and after inhibition of proteasome-mediated degradation. ( a ) SETD2 and H3K36Me3 levels as detected by western blotting in HMC-1.1 and HMC-1.2 cells compared to K562 cells. ( b and c ) Effect of proteasome inhibition by bortezomib on SETD2 and H3K36Me3 levels in HMC-1.1 and HMC-1.2. One of three independent experiments is shown in all panels.

Article Snippet: Assays were performed in triplicate on the ABI 7900HT system (Thermo Fisher Scientific) using pre-designed TaqMan Gene Expression Assays (Thermo Fisher Scientific) for SETD2 (Hs01014784_m1) and TBP (Hs00427621_m1) as control gene.

Techniques: Inhibition, Western Blot

Journal: Leukemia

Article Title: SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

doi: 10.1038/leu.2017.183

Figure Lengend Snippet: Ubiquitinated and SUMOylated SETD2 after proteasome inhibition by bortezomib in HMC-1 cells ( a ) and in SM patients ( b ). Co-immunoprecipitation (IP) with an anti-SETD2 antibody and immunoblotting (western blotting) with anti-ubiquitin and anti-SUMO antibodies performed before and after bortezomib treatment (10 n M for 24 h). H3K36Me3 levels were also assessed by WB. H3 and beta-actin were used as controls. The two ISM patients had near-normal SETD2 protein levels (90.5% and 75.8% of SETD2 expression with respect to a pool of healthy donors); the MCL patient had no SETD2 protein expression (see for SETD2 protein level estimates as assessed by densitometric analysis of western blots). One of three independent experiments is shown in all panels.

Article Snippet: Assays were performed in triplicate on the ABI 7900HT system (Thermo Fisher Scientific) using pre-designed TaqMan Gene Expression Assays (Thermo Fisher Scientific) for SETD2 (Hs01014784_m1) and TBP (Hs00427621_m1) as control gene.

Techniques: Inhibition, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Immunoblot analysis showing localization of SETD2 in whole-cell extracts (WCE) and both nuclear and cytoplasmic (Cyto) compartments of 786-0 and HEK293T cells. Lamin A/C and lactate dehydrogenase (LDH) are used as controls for the nuclear and cytoplasmic fractions, respectively. ( B ) Immunoblot analysis showing coimmunoprecipitation of endogenous SETD2 and endogenous actin in 786-0 cells using SETD2 antibodies from two different sources. ( C ) Immunoblot analysis showing that actin is methylated in SETD2-proficient but not in SETD2-deficient 786-0 cells by immunoprecipitation (IP) of endogenous actin using two different antibodies directed against the SETD2 trimethyl-lysine epitope. Data in (A to C) are representative of experiments repeated at least three times with similar results. ( D ) Deconvolution microscopy imaging of filamentous (F)–actin using phalloidin (green) and SETD2 methyl epitope–specific antibody (magenta) showing colocalization of the methyl mark with F-actin in 786-0 cells. Scale bars, 10 μm. ( E ) Representative intensity profiles of the staining observed with the methyl-specific antibody and phalloidin. Merged images from (D) are shown on the left to indicate the position for line profiles. ( F ) Quantification of intensity profiles seen in (E). y axis represents the ratio of methyl antibody to phalloidin intensity. Each small circle represents a single cell. Large circles represent means from 20 cells for each independent biological replicate; data are means ± SEM from these values ( n = 3).

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Western Blot, Methylation, Immunoprecipitation, Microscopy, Imaging, Staining

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Autoradiography showing in vitro methylation of actin using tritiated S -adenosylmethionine ( 3 H-SAM) as methyl group donor and recombinant glutathione- S -transferase (GST)–tagged SETD2 catalytic SET domain (amino acids 1418 to 1714). Film shows automethylation of SETD2 and histone methylation as positive control. Data are representative of experiments repeated at least three times, with similar results. ( B ) Fluorescence-based in vitro methylation using recombinant tSETD2 (amino acids 1418 to 2564) with purified cardiac muscle (red), smooth muscle (green), skeletal muscle (purple) actins, and recombinant actin (rActin) from HEK293T cells (blue) or E. coli (orange). Data are means ± SEM ( n = 2). ( C ) Fluorescence-based in vitro methylation of actin using recombinant tSETD2 (amino acids 1418 to 2564) following addition of Latrunculin A (LatA) at indicated concentrations. Methyltransferase activity calculated by slope of linear regression from fluorescence trace (a.u./min), against LatA concentration on x axis. Data are means ± SEM ( n = 3). y axis in (B) and (C) plotted after subtracting automethylation from samples with SETD2 alone. ( D and E ) Immunoblot analysis showing recognition of actin proteins by SETD2 methyl epitope antibodies anti-Me3 K40 (D) and anti-Me3 K36 (E) following in vitro methylation with recombinant GST-tagged SETD2 (amino acids 1418 to 1714). Data are representative of experiments repeated at least three times with similar results.

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Autoradiography, In Vitro, Methylation, Recombinant, Positive Control, Fluorescence, Purification, Activity Assay, Concentration Assay, Western Blot

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Amino acid sequence showing conserved KxP SETD2 recognition motif present in all actin isoforms. Position of the lysine residue varies depending on actin isoform; reference to this site as “ActK68” is based on its position in β-actin (ACTB). Histone H3 sequence containing the KxP motif is shown below for reference. ( B ) Representative tandem mass spectrometry (MS/MS) spectrum of trimethylated ActK68 peptides recovered from SETD2-proficient 786-0 cells. m / z , mass/charge ratio. ( C ) Fluorescence-based in vitro methylation assay showing in vitro methylation of biotin-labeled K68-containing actin peptides (amino acids 62 to 78) with recombinant tSETD2 (amino acids 1418 to 2564). Sequence for the peptides used is shown in (A). Data are means ± SEM ( n = 4). ( D ) Immunoblot analysis showing dependency of the ActK68me3 mark on SETD2 by IP of endogenous actin from whole-cell extracts of SETD2-proficient and SETD2-deficient 786-0 cells using the anti-Me3 K68 antibody. Data are representative of experiments repeated at least three times with similar results.

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Sequencing, Residue, Mass Spectrometry, Tandem Mass Spectroscopy, Fluorescence, In Vitro, Methylation, Labeling, Recombinant, Western Blot

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Immunoblot analysis showing decreased F-actin in SETD2-deficient 786-0, HEK293T, and MEF cells. Whole-cell lysate shows absence of SETD2, associated with the expected loss of histone H3K36me3 methylation. ( B ) Quantitation of F-/G- actin ratio (top) and whole-cell lysate actin (bottom) from the data is shown in (A). Data are means ± SEM ( n = 3 for 786-0 and HEK293T; n = 6 for MEF). ( C ) Immunoblot analysis of F-/G-actin ratio in HEK293T cells expressing wild-type or K68A/R mCherry–β-actin. ( D ) Immunoblot analysis of whole-cell lysates shows no change in total actin levels or SETD2 histone methylation with expression of K68A/R mCherry–β-actin. ( E ) Quantitation of F-/G-actin and total actin shown in (C) and (D), respectively. Data are means ± SEM ( n = 3).

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Western Blot, Methylation, Quantitation Assay, Expressing

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: Immunoblot (IB) analysis ( A ) and quantitation ( B ) showing changes in actin polymerization (F-actin in the insoluble fraction) following washout after treatment with the actin depolymerizing agent latrunculin A (LatA). Data are means ± SEM ( n = 4). ( C ) IB analysis using anti-Me3 K68 antibody showing that ActK68me3 occurs on endogenous actin from the insoluble F-actin fraction of SETD2-proficient 786-0 cells; tubulin (which is also methylated by SETD2) is not found in this fraction. Data are representative of experiments repeated at least three times with similar results. Absence of H3K36me3 in the insoluble fraction is used as a control to confirm loss of SETD2 in (A) and (C). ( D ) IP of actin using anti-Me3 K68 antibody from soluble (G-actin) and insoluble (F-actin) fractions of SETD2-proficient cells treated with LatA for indicated times. Light and dark exposures are shown for contrast in amount of methylated actin between fractions. Data are representative of three experiments performed using anti-Me3 K68 and anti-Me3 Pan antibodies with similar results. In an additional experiment performed shortly after cells were brought up from cryopreservation, we saw a less marked loss of methylated insoluble actin with LatA, but saw no increase in methylated soluble actin, consistent with data shown here.

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Western Blot, Quantitation Assay, Methylation, Control

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) In vitro scratch assay at 0 and 24 hours after wound inflection, illustrating that SETD2-deficient cells migrate slower than SETD2-proficient 786-0 cells. Scale bars, 1000 μm. ( B ) Quantification of scratch assays seen in (A). Small circles represent each independent measurement across all experiments. Large circles represent mean from a minimum of nine individual measurements for each independent biological replicate ( n = 4).

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: In Vitro, Wound Healing Assay

Journal: Science Advances

Article Title: The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

doi: 10.1126/sciadv.abb7854

Figure Lengend Snippet: ( A ) Schematic diagram showing interaction between the SETD2 C-terminal and HTT N-terminal proline-rich region (PRR), as reported in ( , ). ( B ) Immunoblot (IB) analysis showing coimmunoprecipitation of mCherry-tagged SETD2 C-terminal [SETD2-(C)-mCherry] and endogenous HTT in HEK293T cells. ( C ) IB analysis showing that actin methylation by SETD2 is dependent on both HTT and HIP1R using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) to immunoprecipitate actin from 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Input lysates shown to confirm knockdown of HTT and HIP1R. ( D and E ) IB analysis (D) and quantitation (E) of decreased insoluble F-actin in 786-0 cells after siRNA-mediated knockdown of HTT or HIP1R . Data are means ± SEM ( n = 3). ( F ) Quantitation of migration data after siRNA-mediated knockdown of HTT or HIP1R in SETD2-proficient versus SETD2-deficient 786-0 cells. Small circles each represent an independent measurement from all biological replicates. Large circles represent mean from 12 measurements for each independent biological replicate ( n = 4). ( G ) IB analysis showing decreased actin methylation after expression of a mutant HTT protein by IP of actin using a SETD2 methyl-epitope antibody (anti-Me3 K36 ) from HEK293T cells expressing a doxycycline (Doxy)–inducible CFP-tagged N-terminal HTT construct containing 94 polyglutamine repeats (CFP-HTT94Q) and a retrotransactivator (rtTA3). Input lysate confirms expression of CFP-tagged mutant protein and loss of SETD2 methylation activity (H3K36me3) without change in endogenous SETD2 expression. Data in (B), (C), and (G) are representative of experiments repeated three times with similar results.

Article Snippet: To identify the interaction between endogenous SETD2 and mCherry-tagged, exogenous actin, SETD2 was immunoprecipitated using whole-cell extracts from HEK293T cells expressing mCherry-ACTB (Addgene plasmid no. 54966).

Techniques: Western Blot, Methylation, Knockdown, Quantitation Assay, Migration, Expressing, Mutagenesis, Construct, Activity Assay

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: (A) Lysates were harvested from undifferentiated human foreskin keratinocytes (HFKs), HFKs stably maintaining HPV16 (HFK-16) or HPV31 (HFK-31) genomes as well as HPV31 positive CIN612 cells, and western blot analysis was performed using an antibody to SETD2. GAPDH served as loading control. (B) Total RNA was extracted from undifferentiated HFKs, HFK-31, HFK-16 and CIN612 cells and quantitative RT-PCR was performed using primers specific to SETD2. Fold change was calculated using 2 −ΔΔCT method. Shown is the fold-change relative to HFKs, which is set to 1. The values represent the average of three independent experiments. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. (C) Lysates were harvested from undifferentiated (T0) HFKs and CIN612 cells, as well as after differentiation in high calcium medium (48, 96hr), and western blot analysis was performed using an antibodies to SETD2 as well as involucrin and keratin 10 (K10) as differentiation controls. p84 serving as a loading control,. (A, C) Shown is a representative image of three independent experiments. Ca = calcium.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: Whole cell lysates were harvested from (A) uninfected HFKs, HFKs retrovirally transduced and stably expressing either wild-type E7 or E7 containing a deletion of the Rb binding domain ( Δ LHCYE) as well as (B) HFKs, HFKs stably maintaining wild-type HPV31 genomes (HFK-31) or genomes containing the E7 Δ LHCYE mutation (HFK-31 Δ LHCYE). Western blot analysis was performed using antibodies to SETD2, (A) p53 and (B) pRb, with p84 as a loading control. Shown is a representative image of three independent experiments. (C) HFKs, HFKs containing wild-type HPV31 genomes (HFK-31) or genomes containing a mutation in E7’s Rb binding domain ( Δ LHCYE), as well as (D) HFKs and CIN612 were treated with 50 ug/ml cycloheximide over an 8hr time course. Lysates were harvested at the indicated time points and western blot analysis was performed using an antibody to SETD2, as well as p84 as a loading control. Graphed are the relative protein levels at each time point normalized to GAPDH, with T0 for each cell line set to 100. Densitometry was performed using Biorad ImageLab 5.0 software. Data shown is representative of three independent experiments.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Stable Transfection, Expressing, Binding Assay, Mutagenesis, Western Blot, Software

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: (A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Transduction, shRNA, Southern Blot, Western Blot, Expressing, Selection, Software

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: (A, B) Chromatin and protein were harvested from CIN612 cells that were undifferentiated (T0) or differentiated in high-calcium medium for 72hr. (A) Chromatin immunoprecipitation (ChIP) was performed using an antibody to H3K36me3 or H3.1 followed by quantitative PCR using 17 primer pairs across the HPV31 genome . To control for differences in viral copy number, PCR data was normalized to input values quantified in parallel for each experiment. The average fold change is graphed and the enrichment is expressed as percent input relative to the first primer set, which is set to one. Averages shown are representative of three independent experiments. Error bars represent means +/- standard error. (B) Western blot analysis was performed using antibodies to SETD2 and to involucrin and K10 as differentiation controls. p84 served as a loading control. Ca = calcium.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: (A) Schematic of the HPV31 genome showing the location of the primer pairs utilized for ChIP analysis. (B, C) Chromatin and protein were harvested from CIN612 cells transduced with either control shRNA or SETD2 shRNA #2 for 72hr (T0), as well as after differentiation in high calcium medium for 72hr. ChIP was performed using an antibody to (B) H3K36me3 or (C) H3.1 using the indicated primer pairs across the HPV31 genome (primer sequences listed in ). Data of ChIP signals from three independent experiments were normalized to 1% of input used. Shown in the fold change in H3K36me3 or H3.1 binding relative to the first primer set, which is set to one. Error bars represent means ± standard error. Statistics were assayed using a student’s t test. * p≤ .05 and ** p≤ .01. (D) Western blot analysis was performed using antibodies to SETD2, to involucrin and K10 as differentiation controls and p84 as a loading control. Ca = calcium.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Transduction, shRNA, Binding Assay, Western Blot

Journal: PLoS Pathogens

Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

doi: 10.1371/journal.ppat.1007367

Figure Lengend Snippet: (A) Schematic of the HPV31 genome showing 5’ splice donor (SD) (closed circles) and 3’ splice acceptor (SA) (open circles) sites. Open reading frames are indicated by the shaded boxes, and promoters are indicated by the arrows. The early (pAE) and late (pAL) polyadenylation sites are indicated at the end of the E5 and L1 ORFs, respectively. (B) CIN612 cells were transiently transduced with lentivirus expressing either a control shRNA (shScram) or SETD2 shRNA #2. 72hr post-transduction, cells were harvested as a T0 (undifferentiated) or differentiated in high calcium medium for 72hr. At each time point, RNA and protein were harvested. (B) End-point PCR amplification was performed (35 cycles) using a 5’ primer in the E7 ORF (nt 766) and a 3’ primer in the L1 ORF (nt 6595), as well as primers specific to GAPDH. Primer sequences are listed in . PCR products were gel purified and sequenced. Shown is a graphical representation of the identified products. Relative levels of L1a and L1b were determined by densitometry using ImageJ software. Values shown represent the ratios of L1a/L1b at each time point. (C) PCR amplification was performed for 25 cycles using the indicated primers to the early region as well as primers specific to GAPDH. Primer sequences are listed in . PCR products generated using the 121F/3373R (E4R) primer pair were gel purified and sequenced. Shown is a graphical representation of the identified products. Relative levels of E1^E4 and E6E7 were determined by densitometry using ImageJ software and normalized to GAPDH. Shown is the fold difference relative to shScram T0, which is set to 1. (D) Western blot analysis was performed using antibodies specific to SETD2 and GAPDH. Shown are representative images of three independent experiments. Ca = calcium. WB = western blot.

Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

Techniques: Transduction, Expressing, shRNA, Amplification, Purification, Software, Generated, Western Blot

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: (A) Graphical representation of the approximate positions of candidate genes within the 3p21.1-p21.3 region of human chromosome 3. Genomic coordinates of all candidate genes were obtained from the National Centre of Biotechnology Information (NCBI) database. (B) Candidate gene copy number (CN) variation analysis of HMEC strains and a panel of nine breast cancer cell lines. (C) Real-time qPCR mRNA expression analysis for SETD2, PBRM1, BAP1 and PARP-3 in breast cancer cell lines and normal breast cells (HMEC’s).

Article Snippet: SETD2, BAP1, PARP-3 and PBRM1 CNV analysis was performed using pre-designed Taqman ® Copy Number Assay primers (Applied Biosystems; Thermo Fisher Scientific, Inc.; assay reference numbers, Hs01027663_cn, Hs02357352_cn, Hs01428519_cn and Hs06624309_cn, respectively).

Techniques: Expressing

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: qRT-PCR analysis of average (Ai) PARP-3 , (Bi) BAP1 , (Ci) SETD2 and (Di) PBRM1 expression levels (mean ± S.E n=3) and (Aii, Bii, Cii, and Dii) pre-spliced hTERT expression levels (mean ± S.E n=3) across five independent stable 21NT-candidate gene transfection clones and five independent stable 21NT-empty vector (EV) clones relative to parental 21NT cells (*p<0.05). Figure 3C shows (iii) SETD2 and (iv) hTERT expression levels (mean ± SD) within 21NT cells 48-144 hours following transient transfection of 21NT cells with pCMVNeo (EV) and pCMVNeo- SETD2 vector constructs, expressed relative to untreated 21NT cells. (NRQ-Normalised Relative Quantity).

Article Snippet: SETD2, BAP1, PARP-3 and PBRM1 CNV analysis was performed using pre-designed Taqman ® Copy Number Assay primers (Applied Biosystems; Thermo Fisher Scientific, Inc.; assay reference numbers, Hs01027663_cn, Hs02357352_cn, Hs01428519_cn and Hs06624309_cn, respectively).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Clone Assay, Plasmid Preparation, Construct

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: Sequences of the synthetic oligonucleotides used as primers for qRT-PCR and thermal cycling parameters

Article Snippet: SETD2, BAP1, PARP-3 and PBRM1 CNV analysis was performed using pre-designed Taqman ® Copy Number Assay primers (Applied Biosystems; Thermo Fisher Scientific, Inc.; assay reference numbers, Hs01027663_cn, Hs02357352_cn, Hs01428519_cn and Hs06624309_cn, respectively).

Techniques: Sequencing