Journal: Cell Biology and Toxicology

Article Title: KMT2D/ZNF460-induced COL9A1-mediated extracellular matrix stiffness maintains the cancer stem cell pool to promote colorectal cancer progression

doi: 10.1007/s10565-025-10053-3

Figure Lengend Snippet: KMT2D binds the COL9A1 enhancer via H3K4me1 modification and recruits ZNF460 to activate COL9A1. ( A ) Analysis of transcriptional regulatory signals around the COL9A1 promoter by Genome Track on the UCSC Genome Browser ( https://genome.ucsc.edu/index.html ). ( B ) The protein expression of ZNF460 and KMT2D in CRC tissues was predicted in UALCAN ( https://ualcan.path.uab.edu/index.html ). ( C ) Prediction of the binding site of ZNF460 on the COL9A1 promoter fragment (chr6:70,278,762–70,279,761) by Jaspar ( https://jaspar.genereg.net/ ). ( D ) The enrichment level of COL9A1 by ZNF460 antibody after knockdown of ZNF460 in HCT 116 cells was examined using ChIP-qPCR. ( E ) The effect of the knockdown of ZNF460 on luciferase activity in HCT 116 cells was examined using a dual-luciferase assay. The enrichment level of COL9A1 by ZNF460 antibody after knockdown of KMT2D ( F ) or overexpression of KMT2D ( G ) by ChIP-qPCR analysis. The level of COL9A1 enhancer fragment enrichment by KMT2D antibody or H3K4me1 antibody after knockdown of KMT2D ( H ) or overexpression of KMT2D (I). KMT2D expression ( J ) and H3K4me1 levels ( K ) in HCT 116 cells after sgRNA transfection were examined using immunofluorescence staining. ( L ) The enrichment level of COL9A1 enhancer by anti-H3 K4 me1 after transfection with sgRNA by ChIP-qPCR analysis. ( M ) COL9A1 transcript levels in HCT 116 cells after sgRNA transfection by RT-qPCR analysis. The results are shown for three biological studies. The bars represent the mean ± SEM; * p <.05, ** p <.01, *** p <.001, and **** p <.0001, as determined by a t-test ( E ) or one-way ANOVA ( J , K , M ) and two-way ANOVA with Tukey’s post hoc test ( D , F , G , H , I , L )

Article Snippet: The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen).

Techniques: Modification, Expressing, Binding Assay, Knockdown, ChIP-qPCR, Luciferase, Activity Assay, Over Expression, Transfection, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: KMT2D/ZNF460-induced COL9A1-mediated extracellular matrix stiffness maintains the cancer stem cell pool to promote colorectal cancer progression

doi: 10.1007/s10565-025-10053-3

Figure Lengend Snippet: Activation of ZNF460 or COL9A1 reverses the inhibitory effect of sh KMT2D on CCSC stemness and proliferative capacity. ( A ) The mRNA expression of KMT2D, ZNF460, and COL9A1 in LRG5 + CCSCs infected with sh KMT2D combined with oe ZNF460 or oe COL9A1 was examined using RT-qPCR. ( B ) The sphere-forming ability of LGR5 + CCSCs after infection was examined using sphere-formation assays. ( C ) The protein expression of CD133 and LGR5 in LGR5 + CCSCs after infection was assessed using Western blot analysis. ( D ) The proportion of Ki67-positive LGR5 + CCSCs after infection was examined using immunofluorescence staining. The results are shown for three biological studies. The bars represent the mean ± SEM; * p <.05, ** p <.01, *** p <.001, and **** p <.0001, as determined by one-way ANOVA with Tukey’s post hoc test

Article Snippet: The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen).

Techniques: Activation Assay, Expressing, Infection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Cell Biology and Toxicology

Article Title: KMT2D/ZNF460-induced COL9A1-mediated extracellular matrix stiffness maintains the cancer stem cell pool to promote colorectal cancer progression

doi: 10.1007/s10565-025-10053-3

Figure Lengend Snippet: KMT2D/ZNF460 axis mediates COL9A1 expression to increase ECM stiffness to promote CCSC stemness and mobility. ( A ) Colonies formed by LGR5 + CCSCs in gels with 2.0 kPa or 20.0 kPa stiffness. ( B ) Spheres formed by LGR5 + CCSCs in gels with 2.0 kPa or 20.0 kPa stiffness. ( C ) The protein expression of Collagen I, LOX, CD133, and LGR5 in LGR5 + CCSCs in gels with 2.0 kPa or 20.0 kPa stiffness was assessed using Western blot analysis. ( D ) The migration and invasion ability of LGR5 + CCSCs in gels with 2.0 kPa or 20.0 kPa stiffness was assessed using the Transwell assay. The results are shown for three biological studies. The bars represent the mean ± SEM; * p <.05, ** p <.01, *** p <.001, and **** p <.0001, as determined by a two-way ANOVA with Tukey’s post hoc test

Article Snippet: The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen).

Techniques: Expressing, Western Blot, Migration, Transwell Assay

Journal: Cell Biology and Toxicology

Article Title: KMT2D/ZNF460-induced COL9A1-mediated extracellular matrix stiffness maintains the cancer stem cell pool to promote colorectal cancer progression

doi: 10.1007/s10565-025-10053-3

Figure Lengend Snippet: Overexpression of COL9A1 potentiates tumorigenesis in AOM/DSS-induced mice. Validation of sh KMT2D lentivirus efficiency ( A ) and sh KMT2D + oe COL9A1 ( B ) lentivirus efficiency in CT26 cells by RT-qPCR. After the euthanasia of C57BL/6 mice, the colon tissues of mice were dissected to analyze tumor load ( C ) and colon length ( D ). ( E ) Inflammatory infiltration of colon tissues was examined using HE staining. ( F ) The extent of collagen deposition in colon tissues was examined using Masson trichrome staining. ( G ) IHC analysis of CD133, LGR5, and Collagen I expression in primary tumors. ( H ) mRNA levels of KMT2D, and COL9A1 in primary tumors were examined using RT-qPCR. The bars represent the mean ± SEM (n = 6); * p <.05, ** p <.01, *** p <.001, and **** p <.0001, as determined by one-way ANOVA with Tukey’s post hoc test

Article Snippet: The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen).

Techniques: Over Expression, Biomarker Discovery, Quantitative RT-PCR, Staining, Expressing

Journal: Cell Biology and Toxicology

Article Title: KMT2D/ZNF460-induced COL9A1-mediated extracellular matrix stiffness maintains the cancer stem cell pool to promote colorectal cancer progression

doi: 10.1007/s10565-025-10053-3

Figure Lengend Snippet: Schematic diagram. KMT2D enhances ECM stiffness in CRC by mediating the H3 K4 me1 modification in the enhancer and recruiting the transcription factor ZNF460 to activate COL9A1 expression, which induces the self-renewal of CCSCs, impairs the immune response, and ultimately promotes the malignant progression of CRC

Article Snippet: The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen).

Techniques: Modification, Expressing

Journal: Journal of molecular biology

Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability

doi: 10.1016/j.jmb.2016.12.016

Figure Lengend Snippet: Yeast Set1 & human Set1-like complexes methylate histone H3K4

Article Snippet: Plasmids pCMV6-MLL4 plasmid (RC221064) expressing FLAG-tagged wild type (WT) full-length human MLL4 was obtained from OriGene.

Techniques:

Journal: Journal of molecular biology

Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability

doi: 10.1016/j.jmb.2016.12.016

Figure Lengend Snippet: (a) Alignment of amino acids in the C-terminal SET domain of human MLL1 (KMT2A) and MLL4 (KMT2D). Tyrosine (Y) residues that are potentially critical for enzymatic activities are highlighted in color. (b – d) 293FT cells were transfected with pCMV6-MLL4 plasmid expressing FLAG-tagged wild type (WT) MLL4 or the Y5426A/Y5512A mutant. (b) Nuclear extracts and histone extracts were analyzed by Western blot using antibodies indicated on the left. The arrow indicates the expected position of MLL4 protein. (c) Anti-FLAG M2 agarose was used to immunoprecipitate (IP) from nuclear extracts. The immunoprecipitates were analyzed by Western blot. (d) In vitro histone methyltransferase (HMT) assay. Proteins immunoprecipitated by anti-FLAG M2 agarose from nuclear extracts of 293FT cells expressing FLAG-tagged WT or mutant MLL4 were subjected to HMT assay using recombinant histone H3 as substrate, followed by Western blot.

Article Snippet: Plasmids pCMV6-MLL4 plasmid (RC221064) expressing FLAG-tagged wild type (WT) full-length human MLL4 was obtained from OriGene.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, In Vitro, HMT Assay, Immunoprecipitation, Recombinant

Journal: Journal of molecular biology

Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability

doi: 10.1016/j.jmb.2016.12.016

Figure Lengend Snippet: (a) Upper panel shows the domains in human and mouse MLL4 proteins. Lower panel shows the alignment of amino acids in the C-terminal SET domain of human and mouse MLL4. Tyrosine (Y) residues that are potentially critical for enzymatic activity are highlighted in color. (b) 293FT cells were transfected with pCMV6-MLL4 plasmid expressing FLAG-tagged WT human MLL4 or the Y5426A/Y5472A/Y5512A mutant. Nuclear extracts and histone extracts were analyzed by Western blot using antibodies indicated on the left. The arrows indicate the expected position of MLL4 protein. (c) Schematic representation of mouse Mll4 WT, targeted and enzyme-dead knock-in (KI) alleles. Deletion of neo selection cassette from the targeted allele by FLP recombinase generates the KI allele. The KI allele carries Y5477A/Y5523A/Y5563A, represented by the red asterisks. The locations of PCR genotyping primers P1 and P2 are indicated by arrows. (d) PCR genotyping of ES cell lines using P1 and P2 primers. (e) Genotyping results from crossing Mll4+/KI with Mll4+/KI mice. The expected ratio of the three genotypes is 1:2:1. (f) Representative images of E9.5~10.5 embryos. (g) Body weights of 3-week-old Mll4+/KI and Mll4+/+ mice (n = 3 for female and n = 6 for male). Data are presented as mean ± SD and P value by Student’s t-test.

Article Snippet: Plasmids pCMV6-MLL4 plasmid (RC221064) expressing FLAG-tagged wild type (WT) full-length human MLL4 was obtained from OriGene.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Knock-In, Selection

Journal: Journal of molecular biology

Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability

doi: 10.1016/j.jmb.2016.12.016

Figure Lengend Snippet: (a – d) Characterization of f/f (Mll4f/f), KI/KI (Mll4KI/KI) and KO (Mll4−/−) ES cell lines. (a) Histone extracts prepared from two different cell lines (#1 and #2) for each genotype were analyzed by Western blot using antibodies indicated on the left. (b) Nuclear extracts were incubated with MLL4 antibody. The immunoprecipitates were analyzed by Western blot. The arrow indicates the expected position of MLL4 protein. (c) qRT-PCR of Mll4 and UTX mRNA expression in cells. (d) Cells were treated with 10 μM MG132 for 1 h and the nuclear extracts were analyzed by Western blot.

Article Snippet: Plasmids pCMV6-MLL4 plasmid (RC221064) expressing FLAG-tagged wild type (WT) full-length human MLL4 was obtained from OriGene.

Techniques: Western Blot, Incubation, Quantitative RT-PCR, Expressing

Journal: Journal of molecular biology

Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability

doi: 10.1016/j.jmb.2016.12.016

Figure Lengend Snippet: (a – d) FLAG-tagged histone H3.3, either WT or K4M mutant, was ectopically expressed in ES cell lines using a lentiviral vector. (a) To analyze the cell growth, 5 × 105 cells were plated at day 0 and the cumulative cell numbers were determined every day for 5 days. (b) Histone extracts from two different cell lines (#1 and #2) were analyzed by Western blot. The arrows indicate the expected position of FLAG-tagged H3.3. (c) Nuclear extracts were analyzed by Western blot. The arrows indicate expected positions of MLL3 and MLL4. The asterisk indicates a non-specific band. (d) qRT-PCR of Mll4, Mll3 and UTX mRNA expression in cells.

Article Snippet: Plasmids pCMV6-MLL4 plasmid (RC221064) expressing FLAG-tagged wild type (WT) full-length human MLL4 was obtained from OriGene.

Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing

Journal: Molecular cell

Article Title: Mll3 and Mll4 facilitate enhancer RNA synthesis and transcription from promoters independently of H3K4 monomethylation

doi: 10.1016/j.molcel.2017.04.018

Figure Lengend Snippet: Key Resources Table

Article Snippet: Rabbit polyclonal anti-Mll4 , Diagenode , Cat#C15310100.

Techniques: Virus, Recombinant, Lysis, Multiplex Assay, DNA Library Preparation, Sequencing, CRISPR, Plasmid Preparation, Software