anti mll4  (Novus Biologicals)

Journal: Nature

Article Title: Bidirectional histone monoaminylation dynamics regulate neural rhythmicity

doi: 10.1038/s41586-024-08371-3

Figure Lengend Snippet: a , LC-MS/MS quantification of H3K4 methylation states (H3K4me0 vs . H3K4me1 vs . H3K4me2; H3K4me3 signal was negligible and was thus omitted) on H3 (1-21) unmodified vs . H3Q5ser vs . H3Q5his peptides, titrating the concentration of MLL1 complex in the system. b – g , Enzymatic quantifications related to Fig. : LC-MS/MS quantification of H3K4 methylation states (H3K4me0 vs . H3K4me1 vs . H3K4me2 vs . H3K4me3) on H3 (1-21) unmodified vs . H3Q5his peptides for (b) MLL1, (c) MLL2, (d) MLL3, (e) MLL4, (f) SETD1A and (g) SETD1B complexes ( n = 3 per peptide/complex). Two-way ANOVA (main effects of interaction: MLL1 – p < 0.0001, F 3,16 = 105.5; MLL2 – p = 0.0002, F 3,16 = 12.29; MLL3 – p < 0.0001, F 3,16 = 86.82; MLL4 – p = 0.0003, F 3,16 = 11.26; SETD1A – p < 0.0001, F 3,16 = 18.86; SETD1B – p < 0.0001, F 3,16 = 41.49), Sidak’s MC tests; significant post hoc comparisons are noted [MLL1: ** p = 0.0017 (H3K4me0), **** p < 0.0001 (H3K4me1/2); MLL2: ** p = 0.0036 (H3K4me0), ** p = 0.0014 (H3K4me1); MLL3: **** p < 0.0001 (H3K4me0 and H3K4me1); MLL4: *** p = 0.0001 (H3K4me1); SETD1A: *** p = 0.0006 (H3K4me0), *** p = 0.0001 (H3K4me1); SETD1B: **** p < 0.0001 (H3K4me0/1)]. h , H3 (1-21) unmodified peptide IPs against recombinant core members of the MLL1 complex (ASH2L, RBBP5, DPY30, MLL1 SET and, WDR5) demonstrating that only WDR5 interacts with the unmodified H3 tail in monomeric form. i , H3 (1-21) unmodified vs . H3Q5his vs . H3Q5ser peptide IPs (Streptavidin) against the recombinant MLL1 complex, followed by western blotting for WDR5 ( n = 3 per peptide). Streptavidin was used to visualize the peptides post-IP, which were used for normalization. One-way ANOVA ( p = 0.0014, F 2,6 = 24.03), Tukey’s MC test: significant comparisons are noted (H3 unmodified vs . H3Q5ser, p = 0.0065; H3Q5his vs . H3Q5ser, p = 0.0013). # p = 0.0462 indicates a significant difference via an a posteriori unpaired Student’s t-test (t 4 = 2.854). Data presented as mean +/– SEM. A.U., arbitrary units, normalized to respective controls (e.g., H3 unmodified peptide). Supplementary Fig. = uncropped blots. Source data provided as a Source Data file .

Article Snippet: H3K4 methyltransferase assays were conducted, unless otherwise indicated, by combining 1.2 μM of either MLL1 (Active Motif, 31423), MLL2 (Active Motif, 31498), MLL3 (Active Motif, 31478), MLL4 (Active Motif, 31499), SETD1A (Active Motif, 81341) or SETD1B (Active Motif, 81342) complexes with 10 μM histone H3 peptide (unmodified versus H3Q5his; 1–21) and 100 μM S -adenosyl methionine in 50 mM Tris, pH 7.5, 50 mM KCl, 5 mM DTT, 5 mM MgCl 2 and 5% glycerol at 25 °C for 3 h. The reactions were quenched by the addition of HPLC solvent A (H 2 O + 0.1% TFA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Methylation, Concentration Assay, Recombinant, Western Blot

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Fiber CSA and fiber type composition in adult Mll4 WT and Mll4 ΔMCK mice. ( A - B ) Percentage of myofibers within each indicated range of CSA in TA muscle of Mll4 ΔMCK and Mll4 WT mice. n = 3 mice for each genotype. ( C ) Representative image of soleus muscles immunolabeled with anti-MyHC1 (green) and anti-laminin (red). Scale bar, 500 μm. ( D , F ) The fiber number of whole TA muscles from Mll4 WT and Mll4 ΔMCK mice of both males and females. n = 3 mice for each genotype. ( E , G ) The percentage of slow fiber among total fiber of soleus muscles from Mll4 WT and Mll4 ΔMCK mice of both males and females. n = 3 mice for each genotype. Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Muscles, Immunolabeling

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Altered myofiber phenotype of 8-week-old Mll4 ΔMCK mice. ( A ) Representative image of the isolated single myofiber. DAPI staining was applied to visualize nuclei. Scale bar, 500 μm. ( B , E ) Myofiber length, ( C , F ) myonuclei accretion, and ( D , G ) myonuclear density were quantified. ( H ) Immunocytochemistry of isolated myofibers of 4-week-old Mll4 ΔMCK and Mll4 WT mice with DAPI (blue) and anti-Pax7 (red). MuSCs are marked with arrowheads. Scale bars, 100 μm. ( I , J ) Pax7 + MuSC number per fiber of Mll4 WT and Mll4 ΔMCK mice of both genders. ( B - G , and I - J ) n = 3 mice for each genotype; >20 fibers per mouse was quantified. Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Isolation, Staining, Immunocytochemistry

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: MuSC depletion due to increased population of differentiating myoblasts in 4-week-old Mll4 ΔMCK muscles. ( A ) Pax7 + MuSC number per 100 fibers was quantified in TA muscles of 0, 2, 4, and 8-week-old Mll4 ΔMCK and littermate control mice. Immunohistochemistry on TA ( B ) and soleus ( C ) muscle section with DAPI (blue), anti-laminin (green), and anti-Pax7 (red). Scale bars, 20 μm. ( D ) Pax7 + MuSC number per 100 fibers was quantified in soleus muscles of 4 and 8-week-old Mll4 ΔMCK and littermate control mice. ( E ) Immunohistochemistry on 4 W TA muscle section with DAPI (blue), anti-Ki67 (green), and anti-Pax7 (red). Scale bars, 20 μm. ( F ) Pax7 + Ki67 + cell number per total Pax7 + cells of 4 W TA muscle. ( G ) Immunocytochemistry of sorted MuSCs with DAPI (blue), anti-Pax7 (red), anti-MyoD (green), and EdU (white – pseudo color for Alexa Fluor 647). MyoD + cells and EdU + cells are marked with arrowheads and sharps, respectively. Scale bars, 20 μm. For enlarged images, Scale bars represent 10 μm. ( H ) MyoD + cell number per total Pax7 + cells. ( I ) Schematic diagram of EdU treatment. ( J ) MyoD + cell number per EdU + Pax7 + cells. ( A , D , F ) n = 3–4 mice for each genotype. ( H , J ) n = 3–4 mice for each genotype; >200 sorted MuSCs per mouse were quantified. ( A , D , F , H , J ) Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Muscles, Control, Immunohistochemistry, Immunocytochemistry

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Induced deletion of Mll4 in the adult stage does not impact myofiber maintenance. ( A ) Schematic diagram of mouse preparation. ( B ) qRT-PCR analysis of myofibers to confirm the downregulation of the Mll4 gene in + 2 W Mll4 ΔHSA mice. ( C ) Percentage of myofibers within each indicated range of CSA, ( D ) gross fiber number, and ( E ) percentage of MyHC2x fibers in TA muscle of Mll4 WT and + 2 W Mll4 ΔHSA mice. ( F ) Gross fiber number and ( G ) percentage of MyHC1 fibers in soleus muscle of Mll4 WT and Mll4 ΔHSA mice. ( H ) Percentage of myofibers within each indicated range of CSA, ( I ) gross fiber number, and ( J ) percentage of MyHC2x fibers in TA muscle of Mll4 WT and + 4 W Mll4 ΔHSA mice. ( K ) Gross fiber number ( L ) and percentage of MyHC1 fibers in soleus muscle of Mll4 WT and + 4 W Mll4 ΔHSA mice. ( B - L ) n = 3 mice for each genotype. Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Quantitative RT-PCR

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Severe MuSC deprivation in Mll4 deleted adult myofibers. ( A ) Immunohistochemistry on TA muscle section with DAPI (blue), anti-laminin (green), and anti-Pax7 (red). ( B ) Pax + MuSC number per 100 fibers of Mll4 WT and Mll4 ΔHSA TA and ( C ) soleus muscle. Scale bars, 20 μm. ( D ) Immunohistochemistry on TA muscle section with DAPI (blue), anti-Ki67 (green), and anti-Pax7 (red). Scale bars, 20 μm. ( E ) Pax7 + Ki67 + cell number per total Pax7 + cells. ( F ) Immunocytochemistry of sorted MuSCs with DAPI (blue), anti-Pax7 (red), anti-MyoD (green), and EdU (white – pseudo color for Alexa Fluor 647). MyoD + cells and EdU + cells are marked with arrowheads and sharps, respectively. Scale bars, 20 μm. For enlarged images, Scale bars represent 10 μm. ( G ) MyoD + cell number per total Pax7 + cells. ( H ) Schematic diagram of EdU treatment. ( I ) MyoD + cell number per EdU + Pax7 + cells, and (J) MyoD + cell number per EdU − Pax7 + cells were quantified. ( K ) Immunohistochemistry on TA muscle section with DAPI (blue), EdU (green), and anti-dystrophin (red). Scale bars, 20 μm. For enlarged images, Scale bars represent 10 μm. ( L ) The number of fiber incorporated EdU + cells per 100 fibers. ( B , C , E , and L ) n = 3–4 mice for each genotype. ( G , H - I ) n = 3–4 mice for each genotype; >200 sorted MuSCs per mouse were quantified. ( B , C , E , L , G , H - I ) Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Immunohistochemistry, Immunocytochemistry

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Defective muscle regeneration capacity after injury in Mll4 deleted TA muscles. ( A ) Schematic diagram of muscle injury and mouse preparation. ( B ) Muscle mass of TA, EDL, GA, and soleus muscles of Mll4 WT − inj and Mll4 ΔHSA−inj mice. ( C ) Representative image of TA muscle labeled with anti-laminin (green) and Hoechst 33,342. Scale bars, 100 μm. ( D ) Percentage of myofibers within each indicated range of CSA in TA muscle of Mll4 WT − inj and Mll4 ΔHSA−inj mice. ( B and D ) n = 3 mice for each genotype. Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Muscles, Labeling

Journal: Skeletal Muscle

Article Title: Mll4 in skeletal muscle fibers maintains muscle stem cells

doi: 10.1186/s13395-024-00369-9

Figure Lengend Snippet: Altered Notch ligand expression in Mll4 deleted myofibers, leading to downregulated notch signaling in MuSCs. ( A ) Evaluation of three public databases proved downregulation of Notch ligands in Mll4 -deleted muscle and myocyte. ( B ) qRT-PCR analysis to compare Notch ligand expression in pubertal (4 W) and adult myofibers. ( C - D ) qRT-PCR analysis to quantify mRNA expression of Notch ligands in myofibers of 4-week-old Mll4 ΔMCK mice and adult Mll4 ΔHSA mice. ( E - F ) qRT-PCR of canonical Notch effectors confirmed general downregulation of Notch signaling in Mll4 ΔMCK and Mll4 ΔHSA MuSCs. ( B - F ) n = 3 mice for each genotype. Data are presented as mean ± SEM of biological replicates. Statistical analyses were performed using unpaired t-test with Welch’s correction

Article Snippet: MCK Cre/+ (stock 006475), HSA MerCreMer/+ (stock 031934), and Mll4 f/f (stock 032152) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: MLL4 expression in NASH patients and NASH protection in Mll4 +/− mouse livers. A , MLL4 gene expression analysis in liver tissues from NASH patients was conducted using GEO data sets (GSE48452 and GSE61260) from the NCBI database. Gene expression levels are presented on a log2 scale, comparing normal (n = 25) and NASH (n = 38) samples. B – I , eight-week-old WT and Mll4 +/− male mice were fed a normal diet (ND) or a methionine and choline-deficient diet (MCDD) for 8 weeks (n = 4–6 for each group). B , weakly body weights (g) were measured. MCDD-fed WT and Mll4 +/− mice showed a significant decrease in body weight compared to ND-fed mice. C , liver weights (g) were measured after 8 weeks of ND and MCDD feeding. D , gross morphology of livers from WT and Mll4 +/− mice was observed after 8 weeks of ND or MCDD feeding. E , serum transaminase levels (AST and ALT) were measured in WT and Mll4 +/− mice (n = 4–5 for each group). F , oil red O staining was performed on liver tissues to assess lipid accumulation. G , H & E staining of liver tissues was used to examine histological changes. H , immunofluorescence staining for F4/80 and DAPI was conducted on liver tissues from WT and Mll4 +/− mice-fed ND or MCDD. Scale bars represents 100 μm. I , qRT-PCR was used to analyze the expression of inflammation-associated genes in liver tissues by (n = 3–4 for each group). Results are presented as mean ± SD. Statistical differences were determined by two-sided Student’s t test ( A ) and two-way ANOVA with Tukey’s multiple comparisons test ( B , C , E , and I ). The exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Expressing, Gene Expression, Staining, Immunofluorescence, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: Resistance of Mll4 +/− mice to MCDD-induced liver fibrosis. A , Masson’s trichrome staining was performed on liver tissues to assess fibrosis. B , Picro Sirius Red staining was used to evaluate collagen deposition in liver tissues. C , immunofluorescence (IF) analysis for α–SMA was conducted on liver sections from WT and Mll4 +/− mice-fed ND or MCDD. Scale bars represents 100 μm. D , expression levels of fibrosis-associated genes in liver tissues were measured by qRT-PCR (n = 3–4 for each group). E , treatment of LX2 cells with TGFβ1 (8 ng/ml) for 24 h elevated fibrosis-associated gene expression, whereas MLL4 knockdown reduced their expression, as measured by qRT-PCR. Data represent the mean ± SD from experiments performed in triplicate. Statistical differences were determined using two-way ANOVA with Tukey’s multiple comparisons test. The exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Staining, Immunofluorescence, Expressing, Quantitative RT-PCR, Gene Expression, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: MLL4 target genes in NASH formation. A , heatmap showing differentially expressed MLL4 target genes under NASH-inducing conditions (MCDD-fed WT: n = 4, MCDD-fed Mll4 +/− : n = 3). B , schematic illustrating that a significant portion of MCDD-regulated genes are MLL4-dependent. C , top significant biological functions and upstream regulators of differentially expressed genes in Mll4 +/− livers. The p -value was calculated using a right-tailed Fisher’s Exact test. D , schematic representation showing that a portion of MCDD-induced genes contain ChIP-seq peaks for both MLL4 and p65. E , heatmap clustering of MCDD-induced MLL4 target genes involved in the NF-κB pathway for NASH formation. An asterisk (∗) denotes MLL4 peaks in brown preadipocytes, and a hash (#) denotes p65 peaks in LPS-treated livers. F , co-immunoprecipitation (CoIP) of MLL4 and p65 in HepG2 cells. HepG2 cells expressing Flag-p65 were subjected to immunoprecipitation with an anti-MLL4 antibody, followed by immunoblotting with an anti-p65 antibody. This revealed that MLL4 interacts with p65, and this interaction is enhanced by LPS (100 ng/ml) treatment for 2 h. G , Hepa1c1c7 cells were transfected with NF-κB:luciferase reporter and expression vectors for p65, sh-control, or sh-MLL4. Luciferase activity was normalized to β-galactosidase activity. Transfections were repeated independently at least three times. Data are shown as relative luciferase units (RLU) (mean ± SD). H , qRT-PCR results indicated that Ccl2 , an NF-κB target gene, was induced by LPS (100 ng/ml) treatment for 6 h, but this induction was suppressed by MLL4 knockdown in Hepa1c1c7 cells. The reduced expression of MLL4 by sh-MLL4 was confirmed by qRT-PCR. I , Ccl2 expression in primary hepatocytes from WT mice was induced by LPS (100 ng/ml) treatment for 6 h but not in hepatocytes from Mll4 +/− mice (n = 2 for each group). J , public datasets from the NCBI GEO database ( http://www.ncbi.nlm.nih.gov/geo/ ) were analyzed for gene expression correlation between MLL4 and CCL2 using NASH patient GEO datasets (GSE48452 and GSE61260). The correlation coefficient (r 2 ) was calculated using Pearson’s correlation test. The x- and y-values in Figure 3 J are log2-transformed. The red solid line represents the regression line, and the red dashed line indicates one standard error bounds. Results are presented as mean ± SD. Statistical differences were determined by two-sided Student’s t test ( G ) and two-way ANOVA with Tukey’s multiple comparisons test ( H and I ). Exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: ChIP-sequencing, Immunoprecipitation, Expressing, Western Blot, Transfection, Luciferase, Control, Activity Assay, Quantitative RT-PCR, Knockdown, Gene Expression, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: Mild inhibition of NASH in hepatocyte-specific MLL4-deleted mice. A , eight-week-old Mll4 f/f (Control) (n = 2) and Mll4 f/f ; Albumin -cre ( Mll4 -AKO) (n = 3) mice were fed an MCDD for 4 weeks. B and C , body weight (g) and liver weight (g) of Mll4 f/f (n = 2) and Mll4 -AKO (n = 3) mice after 4 weeks of MCDD feeding. D , gross liver morphology from WT and Mll4 -AKO mice sacrificed after 4 weeks of MCDD. E , IF analysis for F4/80, Masson’s trichrome staining, and Picro Sirius Red staining of liver tissues from Mll4 f/f and Mll4 -AKO mice-fed MCDD. Scale bars represents 100 μm. F and G , expression of pro-inflammatory genes and fibrosis-associated genes in liver tissues from Mll4 f/f and Mll4 -AKO mice-fed MCDD (n = 2–3 for each group). H , expression of Ccl2 in liver tissues from Mll4 f/f and Mll4 -AKO mice-fed ND or MCDD, as measured by qRT-PCR (n = 2 for each group). Results are presented as mean ± SD. Statistical differences were determined by two-sided Student’s t test ( C , F and G ) and two-way ANOVA with Tukey’s multiple comparisons test ( B and H ). The exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Inhibition, Control, Staining, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: D eletion of MLL4 in KCs/macrophages reduces NASH. A , eight-week-old Mll4 f/f (Control) (n = 8) and Mll4 f/f ; LysM -cre ( Mll4 -LKO) (n = 5) mice were fed with MCDD for 4 weeks. Schematic outline of experimental approaches and analysis. B and C , body weight (g) and liver weight (g) of Mll4 f/f (n = 8) and Mll4 -LKO (n = 5) mice after 4 weeks of MCDD feeding. D , representative captured liver tissues of experimental mice at the end of experiments. E , levels of serum transaminases (AST and ALT) measured from Mll4 -LKO (n = 3) and Mll4 f/f mice (n = 3) fed MCDD. F , H & E staining, IF analysis for F4/80, and Sirius Red staining of liver tissues from Mll4 f/f (n = 6) and Mll4 -LKO (n = 3) fed MCDD. Scale bars represents 100 μm. G and H , qRT-PCR analysis for inflammation- and fibrosis-associated genes in liver tissues from MCDD-fed Mll4 f/f (n = 6–7) and Mll4 -LKO (n = 4–5). Results are presented as mean ± SD. Statistical differences were determined by two-sided Student’s t test ( C , E , G and H ) and two-way ANOVA with Tukey’s multiple comparisons test ( B ). The exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Control, Staining, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: Macrophage/monocyte-specific MLL4 deletion inhibits M1 phenotype. A , flow cytometry analysis of primary Kupffer cells (KCs) from Mll4 f/f (n = 3) and Mll4 -LKO (n = 3) mice fed an MCDD for 4 weeks. Bottom: Mean fluorescence intensity (MFI) of CD80 + M1 KCs and CD163 + M2 KCs was measured. B , expression of M1 and M2 markers in primary bone marrow–derived macrophages (BMDMs) from MCDD-fed Mll4 f/f (n = 4–8) and Mll4 -LKO (n = 3–5) mice, determined by qRT-PCR. C , M1 marker expression in primary BMDMs from ND-fed Mll4 f/f (n = 3) and Mll4 -LKO (n = 3) mice treated with LPS (100 ng/ml) for 6 h, assessed by qRT-PCR. Results are shown as mean ± SD. Statistical differences were determined by two-sided Student’s t test ( A and B ) and two-way ANOVA with Tukey’s multiple comparisons test ( C ). The exact p -values are reported in each graph.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Flow Cytometry, Fluorescence, Expressing, Derivative Assay, Quantitative RT-PCR, Marker

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet: Critical roles of MLL4 in NF-κB target gene expression in the liver. A , chromatin immunoprecipitation (ChIP) analysis showing MCDD-enhanced recruitment of p65 and MLL4 to the Ccl2-κB site in primary hepatocytes from WT mice-fed ND or MCDD. B , ChIP results displaying elevated H3K4me1 levels at the Ccl2-κB site in primary hepatocytes from WT livers under MCDD, but not in primary hepatocytes from Mll4 +/− livers. C and D , ChIP for LPS-enhanced recruitment of p65 and MLL4 to the Tnfα-κB and Nos2-κB sites in primary BMDMs from WT mice. E , ChIP showing increased H3K4me1 levels at the Tnfα-κB and Nos2-κB sites in primary BMDMs from Mll4 f/f , but not from Mll4 -LKO. Results are shown as mean ± SD. Statistical differences were evaluated using a two-sided Student’s t test ( A , C , and D ) and two-way ANOVA with Tukey’s multiple comparisons test ( B and E ). The exact p -values are reported in each graph. F , working model: MLL4 plays a key role in NASH development. MCDD or LPS triggers activation of the NF-κB-MLL4 axis, promoting the decoration of NF-κB target genes with H3K4me1/2 marks in the liver. This leads to the transactivation of Ccl2 in hepatocytes, which recruits macrophages to damaged liver sites. In macrophages, MLL4 drives the transcription of inflammatory, fibrotic, and M1 marker genes, exacerbating NASH progression.

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Targeted Gene Expression, Chromatin Immunoprecipitation, Activation Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet:

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The methyltransferase MLL4 promotes nonalcoholic steatohepatitis by enhancing NF-κB signaling

doi: 10.1016/j.jbc.2024.107984

Figure Lengend Snippet:

Article Snippet: To generate hepatocyte and myeloid cell-specific Mll4 -deleted mouse lines ( Mll4 -AKO and Mll4 -LKO), Mll4 f/f mice were crossed with Albumin -Cre or LysM -Cre mice (The Jackson Laboratories), producing Mll4 f/+ ; Albumin -Cre or Mll4 f/+ ; LysM -Cre mice respectively.

Techniques: Sequencing