Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 1 Selection of MEN1 missense mutation-derived forms of menin. A Nine disease-relevant MEN1 missense mutations were selected based on their predicted stability in silico and ability to bind to MLL1 and/or JunD. The predicted effects of the mutations are listed. B The nine MEN1 mutations are projected on the menin structure

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques: Selection, Mutagenesis, Derivative Assay, In Silico

Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 6 Genome-wide binding events of the menin mutants. A Correlation of the coverage between WT menin and menin mutants. Values represent Pearson correlations of normalized coverage calculated for 10-kb bins. B Coverage tracks of the regions bound by menin WT and menin mutants. The left box highlights a genomic region occupied by both menin WT and mutant proteins, while the right box highlights a genomic region where binding of the menin mutants R52G, E255K and E359K was lost. C Percentages of peaks retained at menin WT bound sites by greenCUT&RUN mapping using the indicated menin mutant. In blue, retained binding at menin–JunD sites without MLL1/H3K4me3 is shown. In red, retained binding of mutant forms of menin at WT menin–MLL1/H3K4me3 sites without JunD is shown. D Heatmaps showing coverage on the retained (upper panel) and lost (lower panel) peaks with menin mutants relative to WT menin. The number of the peaks are given on the left side. The coverage is shown on the top of each heatmaps. Please note that the E408Q mutant essentially behaves like WT menin

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques: Genome Wide, Binding Assay, Mutagenesis

Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 7 Projection of the selected MEN1 gene mutations menin R52G, E255K, E359K and E408Q (in green) on the tertiary structure of WT menin. Overlapping positions of the MLL1 peptide (in brown) and of the JunD peptide (in blue) are indicated

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 1. Mll1+/2 mice have an altered response to PPD antigen challenge at 4 d post-PPD bead challenge. (A and B) H&E staining of lungs from WT and Mll1+/2 mice at 4 d postbead injection and a graph quantifying significant differences in granu- loma size. *P , 0.03, as determined by Student’s t test. (C and D) Quantification of IFN-g at protein and mRNA levels. (E) Analysis of infiltrating myeloid cell populations in the lung. Results are representative of 3 separate experiments. *P , 0.007, as determined by Student’s t test. Neutrophils [polymorphonuclear neutrophils (PMN)] were defined as autofluorescent- negative CD11b+CD11c2Ly6G+, DCs as autofluores- cent-negative CD11c+CD11b+Ly6C+MHC II+, and macrophages (MF) as autofluorescent-negative CD11b+F/480+MHC IImidhigh. (F) Analysis of total CD4+ T cells, as well as T cell subsets in the lung and lymph node. Effector CD4 cells were defined as CD44hiCD62Llo, and memory cells were defined as CD44hiCD62L+CCR7+. *P , 0.05, as determined by Student’s t test. (G) Staining of lung sections with antibodies to H3K4Me3 in WT and Mll1+/2 mice. The beads at the center of the granulomas are represented by the dashed circles. (H) Mll1 expression in T cell subsets isolated from the spleen by FACS at 14 d post- PPD immunization. Effector and memory cells were isolated, as described in F. Na¨ıve cells were defined as CD44loCD62L+. Expression of Mll1 in CD4+ effector cells isolated from the lungs of PPD-immunized and -challenged mice at 4 d postinjection. For all data, n = 3–5 animals/experiment. *P , 0.05, as de- termined by Student’s t test. Results are representative of 3 independent experiments.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Injection, Expressing, Isolation

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 2. Mll1+/2 CD4+ T cells cause altered granuloma pathology in Rag22/2 mice. Quantifica- tion of IFN-g protein (A) and Mll1 mRNA expression (B) in the lungs of Rag22/2 mice receiving Mll1+/2

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 3. Mll1+/2 Th1 cells have reduced proliferative capacity and have reduced ex- pression of CCND3. (A) Assessment of pro- liferation of WT and Mll1+/2 T cells under Th0 and Th1 conditions by [3H]thymidine uptake. *P # 0.01, as determined by 1-way ANOVA. (B) Depiction of the ratio of T cells proliferating in the presence (Th1) vs. the absence (Th0) of IL-12. This ratio is shown for WT and Mll1+/2

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 4. STAT4 binds to the Mll1 promoter. (A) PCR data assessing the levels of Mll1 in T cells cultured under normal Th0 or Th1 conditions or Th1 conditions with the addition of 1 mM of the JAK inhibitor tofacitinab (CP690,550). *P # 0.05, as determined by 1-way ANOVA. (B) Assessment of Mll1+

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Culture

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 5. MLL1 binds to genes critical to Th1 cell differentiation. ChIP assay to quantify H3K4Me3 modifications and MLL1 binding to the promoters of Tbx21, Ifng, IL12RB2, and Ccnd3 in murine Th0 and Th1 cells. TSS, Transcription start site. Negative numbers on the abscissa represent kilobases upstream of the TSS. Data are pooled from 3 independent experiments performed with 1.0 3 107 pooled cells from 3 to 5 animals for each replicate. *P # 0.05 by 1-way ANOVA.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Differentiation, Binding Assay

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 6. Mll1 is essential for the T cell recall response. (A) IFN-g production by Mll1+/2 CD4+ T cells in vitro after 5 d of activation and 3 d of rest, followed by an additional 48 h of stimulation with anti- CD3/anti-CD28. *P # 0.03 by Student’s t test. (B) Proliferation of Mll1+/2 T cells in the same conditions as in (A). *P # 0.03 by Student’s t test. (C) IFN-g production from CD4+ T cells isolated from the lungs of PPD-sensitized and -challenged mice that were then cocultured with BMDCs pulsed with PPD for 48 h. **P # 0.001 by Student’s t test. (D) Assessment of pro- liferation and IFN-g production during the tetanus toxoid recall response when cocultures of T cells and monocyte-derived DCs from the same donor were incubated with the Mll1/menin inhibitor MI-2-2 for 5 d in the presence of tetanus toxoid. All comparisons in (D) are made by use of the DMSO control. *P , 0.05 by Student’s t test compared with the 15 mM dose of inhibitor. Culture supernatants were analyzed by bioplex (A and C) or standard sandwich ELISA (D). Proliferation was measured by pulsing with [3H]thymidine on day 4 of culture and measuring uptake over an 18 h period. (A–C) Data are pooled from 3 independent experiments with the pooled cells of 2–3 animals for each replicate.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: In Vitro, Activation Assay, Isolation, Derivative Assay, Incubation, Control, Sandwich ELISA

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a Epigenomics Roadmap indicates that genes with H3K4me3 peaks in their promoter regions are enriched in LFS astrocytes. b Word clouds represent proteins inferred with high confidence to interact with SVIL in LFS astrocytes. c Endogenous interaction between SVIL and MLL1 in LFS-GFP-SVIL astrocytes. d PLA analysis indicates that endogenous mutant p53 forms a complex with SVIL and MLL1 in LFS astrocytes. Scale bar, 10 µm. e Depletion of SVIL impairs p53(G245D)/SVIL/MLL1 complex formation in LFS astrocytes. f Depletion of SVIL impairs mutant p53/SVIL/MLL1 complex formation. Scale bar, 10 µm. g H3K4me3 peaks on the YTHDF2 promoter are reduced upon MLL1 depletion or MLL1 inhibition by OICR-9429 in LFS astrocytes. h RT-qPCR analysis demonstrates decreased YTHDF2 mRNA expression upon MLL1 knockdown in LFS astrocytes but not WT astrocytes ( n = 3 biologically independent samples). i Immunoblotting indicates reduced YTHDF2 protein upon the treatment with MLL1 inhibitors OICR-9429 and MI-2-2 in LFS astrocytes. j OICR-9429 and MI-2-2 selectively inhibit cell proliferation of LFS astrocytes ( n = 5 biologically independent samples). k In vitro AIG assay demonstrates decreased colony numbers upon MLL1 depletion in LFS astrocytes ( n = 5 biologically independent samples). l Ectopic YTHDF2 expression rescues SVIL or MLL1 knockdown-induced growth inhibition in LFS astrocytes ( n = 6 biologically independent samples). m In vitro AIG assay demonstrates decreased colony numbers upon SVIL or MLL1 knockdown that are rescued by YTHDF2 expression ( n = 3 biologically independent samples)). n Colony-forming assay demonstrates that OICR-9429 and MI-2-2 cause more severe growth inhibition of LNZ308-p53(G245D) cells than LNZ308-Vector cells ( n = 3 biologically independent samples). o Images of WT (mCherry + ) and LFS (GFP + ) co-cultured cerebral organoids examined by light sheet fluorescence microscopy. Scale bar, 500 µm. p OICR-9429 selectively inhibits proliferation of the LFS-derived population of WT/LFS co-cultured cerebral organoids ( n = 19 biologically independent samples). The results are representative of at least three independent experiments ( c – f , i , o ). The data are presented as the mean ± SEM; two-way ANOVA with Bonferroni’s multiple comparison test ( h , j – n ); unpaired two-tailed Student’s t test ( p ); *** P < 0.001. ns not significant. Source data and exact P values are provided in the Source Data file.

Article Snippet: Primary antibodies, including anti-p53 antibody (Santa Cruz, sc-126), anti-SVIL antibody (Sigma-Aldrich, S8695), and anti-MLL1 antibody (Novus Biologicals, NBP2-55237), were incubated overnight at 4 °C.

Techniques: Mutagenesis, Inhibition, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, In Vitro, Plasmid Preparation, Cell Culture, Fluorescence, Microscopy, Derivative Assay, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a m 6 A MeRIP-seq indicates distribution of m 6 A peaks in different regions (5’UTR, first exon, other exon, and 3’UTR) of transcripts. Pie chart shows the percentage of m 6 A peaks within distinct regions of transcripts in LFS astrocytes. b Violin plot demonstrates significant elevation of highly m 6 A-marked transcripts upon YTHDF2 depletion. c Examination of YTHDF2 IP enrichment by eCLIP-seq. d Metagene plot of YTHDF2 eCLIP-seq indicates enrichment of YTHDF2-interacting mRNA peaks in the 3’UTR clustered around stop codons. e Motif analysis demonstrates that YTHDF2 binding motifs are similar to the consensus m 6 A motif RRACH. f Venn diagram identifying 84 YTHDF2-targeted m 6 A transcripts validated by a combination of m 6 A MeRIP-seq, YTHDF2 eCLIP-seq, and RNA-seq in LFS astrocytes. These transcripts include CDKN2B and SPOCK2 mRNAs. g IGV track views of m 6 A peaks located on CDKN2B and SPOCK2 transcripts in LFS astrocytes. h RT-qPCR indicates decreased expression of YTHDF2-targeted CDKN2B and SPOCK2 transcripts in LFS astrocytes ( n = 3 biologically independent samples). i Low expression of YTHDF2-targeted CDKN2B and SPOCK2 is correlated with poor overall survival of LGG/GBM patients. Log-rank (Mantel–Cox) test is performed to compute significance. j Immunostaining demonstrates lower CDKN2B in engrafted LFS cerebral organoids (upper panel) and increased CDKN2B in YTHDF2-depleted engrafted LFS cerebral organoids (lower panel), Scale bar, 100 µm. Anti-CDKN2B antibodies only recognize human but not mouse CDKN2B proteins. k mRNA stability assay demonstrates that YTHDF2 knockdown leads to an increased half-life of CDKN2B and SPOCK2 mRNAs. shCtrl-LFS and shYTHDF2-LFS astrocytes are treated with actinomycin D and total RNAs are isolated at 0, 30, and 60 min. ( n = 3 biologically independent samples). l RT-qPCR demonstrates elevated expression of YTHDF2 targets CDKN2B and SPOCK2 upon depletion of p53, YTHDF2, or SVIL as well as inhibition of MLL1 function by OICR-9429 or MI-2-2 in LFS astrocytes ( n = 3 biologically independent samples). The results are representative of at least three independent experiments ( c , j ). The data are presented as the mean ± SEM); two-way ANOVA with Bonferroni’s multiple comparison test ( l ); unpaired two-tailed Student’s t test ( h ); multiple t test ( k ); ** P < 0.01, *** P < 0.001. Source data and exact P values are provided in the Source Data file.

Article Snippet: Primary antibodies, including anti-p53 antibody (Santa Cruz, sc-126), anti-SVIL antibody (Sigma-Aldrich, S8695), and anti-MLL1 antibody (Novus Biologicals, NBP2-55237), were incubated overnight at 4 °C.

Techniques: Binding Assay, RNA Sequencing, Quantitative RT-PCR, Expressing, Immunostaining, Stability Assay, Knockdown, Isolation, Inhibition, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a In vivo mouse xenograft study demonstrates that knockdown of YTHDF2, SVIL, or MLL1 hampers LNZ308-p53(G245D) tumor growth ( n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. b Expression of CDKN2B or SPOCK2 inhibits cell proliferation of LFS astrocytes ( n = 6 biologically independent samples). c In vitro AIG assay demonstrates ectopic expression of CDKN2B or SPOCK2 leading to decreased colony numbers of LFS astrocytes. All colonies are counted and measured after 2-month culture ( n = 6 biologically independent samples). d In vivo mouse xenograft study shows ectopic expression of CDKN2B or SPOCK2 abrogating LNZ308-p53(G245D) tumor growth ( n = 3 biologically independent mice). The sizes of the tumors were measured at the indicated time. e Knockdown of CDKN2B or SPOCK2 rescues YTHDF2 depletion-induced growth inhibition in LFS astrocytes ( n = 6 biologically independent samples). f In vitro AIG assay demonstrates knockdown of CDKN2B or SPOCK2 rescuing in vitro colony formation of YTHDF2-depleted LFS astrocytes. All colonies are counted and measured after 2-month culture ( n = 6 biologically independent samples). g Xenograft study indicates that knockdown of CDKN2B or SPOCK2 rescues YTHDF2 knockdown-induced LNZ308-p53(G245D) tumor growth inhibition in nude mice ( n = 4 biologically independent mice). The sizes of the tumors were measured at the indicated time. h Transcriptome analysis of CDKN2B-restored LFS astrocytes. Bubble plot for visualizing enriched GO and KEGG pathway analyses of differentially upregulated genes in CDKN2B-restored LFS astrocytes. X axis label represents the enrichment factor (number of differentially expressed genes enriched in the pathway/total number of genes in the pathway) and Y axis label represents GO annotation and KEGG pathway. Size and color of the bubble represent number of differentially expressed genes enriched in the GO or KEGG pathways and enrichment significance ( P value), respectively. The data are presented as the mean ± SEM; two-way ANOVA with Bonferroni’s multiple comparison test ( a – g ). * P < 0.05, *** P < 0.001. Source data and exact P values are provided in the Source Data file.

Article Snippet: Primary antibodies, including anti-p53 antibody (Santa Cruz, sc-126), anti-SVIL antibody (Sigma-Aldrich, S8695), and anti-MLL1 antibody (Novus Biologicals, NBP2-55237), were incubated overnight at 4 °C.

Techniques: In Vivo, Knockdown, Expressing, In Vitro, Inhibition, Comparison

Journal: Nature Communications

Article Title: Rewired m 6 A epitranscriptomic networks link mutant p53 to neoplastic transformation

doi: 10.1038/s41467-023-37398-9

Figure Lengend Snippet: a Elevated YTHDF2 expression is observed in LFS stromal cells with a heterozygous M133T mutation but not LFS stromal cells with a heterozygous 12141delG frameshift mutation compared with WT stroma ( n = 3 biologically independent samples). The data are presented as the mean ± SEM; unpaired two-tailed Student’s t test. * P < 0.05. b Box plots of TCGA RNA expression profiles (log2) in TCGA tumors with p53 WT or p53 hotspot missense mutations in LGG, GBM, BRCA, and READ specimens. Two-sided Mann Whitney Wilcoxon test is performed to compute significance. Tumors with a p53 hotspot missense mutation demonstrate decreased p21 and PUMA mRNA expression but elevated Y THDF2 mRNA expression. Box edges delineate lower and upper quartiles, the center line represents the median, and whiskers extend to 1.5 times the interquartile range. c Kaplan–Meier curves compare survival in LGG and GBM specimens with high or low levels of YTHDF2-targeted transcripts. Log-rank (Mantel–Cox) test is performed to compute significance. d Estimated hazard ratios and 95% confidence intervals for TCGA LGG and GBM patients expressing high levels of 20 YTHDF2-targeted genes. High expression of 13 of these 20 genes is positively associated with lower hazard ratios and increased survival with FDR q -value less than 5%. e A model linking the mutant p53/SVIL/MLL1 transcriptional regulatory complex to epitranscriptomic changes driving gliomagenesis. In our proposed model, mutant p53 interacts with SVIL, recruits MLL1 to the YTHDF2 promoter, and then induces YTHDF2 transcription. Elevated YTHDF2 downregulates numerous m 6 A-marked transcripts, including CDKN2B and SPOCK2 , promotes neoplastic transformation and initiates gliomagenesis. MLL1 inhibitors selectively suppress YTHDF2 expression, LFS and mutant p53 cell survival, and neoplastic transformation. Source data and exact P values are provided in the Source Data file.

Article Snippet: Primary antibodies, including anti-p53 antibody (Santa Cruz, sc-126), anti-SVIL antibody (Sigma-Aldrich, S8695), and anti-MLL1 antibody (Novus Biologicals, NBP2-55237), were incubated overnight at 4 °C.

Techniques: Expressing, Mutagenesis, Two Tailed Test, RNA Expression, MANN-WHITNEY, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells

doi: 10.3390/ijms25094704

Figure Lengend Snippet: Decrease in insulin secretion in response to glucose loading in Kmt2a -knockdown βHC-9 cells. ( A ) Kmt2A expression was silenced via siRNA-mediated Kmt2A knockdown. Small interfering RNA targeting Kmt2A (siKmt2A) or control siRNA (Ctl) (200 nM) was introduced into βHC-9 cells via electroporation, and Kmt2A mRNA expression was quantified after 48 h via real-time polymerase chain reaction. ( B ) Glucose-stimulated insulin secretion tests were performed in Kmt2a -knockdown βHC-9 cells. ( C ) Scatter plot of the microarray data of Kmt2A -knockdown βHC-9 cells. ( D ) SLC2a1 and SLC2a2 expression. Data represent the mean ± standard error of mean from triplicate samples. Asterisks indicate significant differences from Ctl-transfected cells. The experiment was repeated twice, with similar results. The white and black columns represent the siClt- and siKmt2A-transfected groups, respectively.

Article Snippet: The TaqMan probes for Gapdh (Mm99999915_g1), Kmt2A (Mm01179235_m1), Slc2a1 (Mm05908127_s1), and Slc2a2 (Mm00446229_m1) were obtained from Applied Biosystems (Bedford, MA, USA).

Techniques: Knockdown, Expressing, Small Interfering RNA, Control, Electroporation, Real-time Polymerase Chain Reaction, Microarray, Transfection

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: a Schematic of KMT2A protein structure (NP_001184033.1), with domains (and motifs) and the location of the identified de novo missense and splice variants. Domains were predicted by SMART program (http://smart.embl-heidelberg.de/). DNA binding AT hooks 1–3 (aa 169–180; aa 217–227; aa 301–309), CXXC domain (aa 1150–1198), zinc finger PHD-type 1–3 (aa 1431–1482; 1479–1533; 1566–1630), TAD domain (aa 2850–2858), bromodomain (aa 1703–1748), FYR N-terminal domain (aa 2021–2077), FYR-C terminal domain (aa 3666–3747), WDR5 interacting motif (aa 3765–3773), SET domain (aa 3832–3948), and post-SET domain (aa 3956–3972). b Sequence analysis of PCR products from the patient’s and parent’s (mother and father) DNA showing the different de novo variants. Asteriks indicate the position of the variants

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Binding Assay, Sequencing

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Facial characteristics of the ID patients carrying a KMT2A variant. Patient 2 (P2) carries the c.8558T>G (p.(Met2853Arg)) variant, Patient 3 (P3) carries the c.3581G>A (p.(Cys1194Tyr)) variant and Patient 4 (P4) carries the c.11322–1G>A variant

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Variant Assay

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: a Sequence analysis of RT-PCR products from the patients (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). In all cases, the two alleles were detected. b KMT2A mRNA expression in controls (WT) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). Y: KMT2A relative expression. Normalization factor was based on the GAPDH reference gene. Each chart represents one KMT2A relative quantification of a stimulated fibroblast culture. Errors bars represent SEM. c Western blot analysis of KMT2A protein of human control (WT1, WT2, WT3, and WT4; WT: all controls) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). GAPDH was used as loading control. Densitometry of western blotting was performed using Image J software. Data in arbitrary units (a.u.) represent the mean ± SEM of four separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between control and KMT2A mutated fibroblasts. d Concerning the patient bearing the potential splice variant, RT-PCR using primers in exon 32 and 35 on cDNA from the patient and controls only showed one distinct band. Sequencing of the RT products showed two transcripts, the wild-type transcript and the mutated transcript with a deletion of the first nucleotide G of exon 33. This variant results in a premature stop-codon and is predicted to produce a truncated protein deleted of its C-terminal SET domain (p.Lys3775Serfs*32). This data suggests a change in the acceptor site of intron 32 which uses the mutated nucleotide A and the first G of the exon 33 to reconstitute the acceptor site

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Software, Variant Assay

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Mapping nuclear targeting signals of wild-type (WT) and mutated KMT2A. Wild type or mutated KMT2A constructs (c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg))) were transiently transfected into COS7 cells and detected by staining with anti-MLL-1 (KMT2A) antibody. a Representative examples of typical patterns; uniform pattern and dot patterns (small dots or bigger patches absent within the nucleoli). b Distribution (% ± SEM) of the different nuclear patterns of cells expressing wild-type or mutated KMT2A constructs. Results were obtained by using data from more than 600 transfected cells of each construct in four independent experiments. The KMT2A c.3460C>T (p.(Arg1154Trp)) mutant abolishes significantly its capability to produce big dots (***χ 2 test with p < 0.0001)

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Construct, Transfection, Staining, Expressing, Mutagenesis

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Expression of target genes of KMT2A (CDKN2C, CDKN1B, SIX2, HOXA9 and MEIS1) in wild-type fibroblasts (WT) and in human fibroblasts bearing different KMT2A variants (patients, c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg)), c.11322–1G>A).**p < 0.001, ***p < 0.0005 between control and KMT2A mutated fibroblasts

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Expressing