Journal: The Journal of Biological Chemistry

Article Title: Colorectal cancer hot spot mutations attenuate the ASXL-MLL4 interaction

doi: 10.1016/j.jbc.2025.108333

Figure Lengend Snippet: Interdependent genomic binding of MLL4 and BAP1 on enhancers. Wild type (WT) and MLL4 knockout (KO) HCT116 were infected with a doxycycline-inducible lentiviral vector expressing BAP1-T7. Cells were treated with 1 mg/ml doxycycline for 24 h and collected for Western blot using indicated antibodies and ChIP-seq of BAP1-T7 using a T7 antibody. WT, MLL4 KO, and BAP1 KO HCT116 were collected for Western blot and ChIP-seq of MLL4. A , Western blot analyses of nuclear extracts of MLL4 KO cells. B , genomic distribution of BAP1-T7 binding regions. Promoters were defined as transcription start sites ± 1kb. Enhancers were defined as H3K4me1 + promoter-distal regions. C and D , Western blots in WT and BAP1 KO HCT116 cells. Whole-cell lysates were analyzed using indicated antibodies (two different anti-BAP1 antibodies, D7W7O and D1W9B, were used). Histone modification levels in ( D ) were quantified and normalized to H3 of BAP1 KO cells. E , Venn diagram depicting genomic binding of BAP1-T7 and MLL4. F–H , genomic binding of MLL4 and BAP1 on enhancers are interdependent. Heat maps ( F ) and average profiles ( G ) for MLL4, BAP1-T7, and H3K4me1 were aligned around the center of MLL4 binding sites on MLL4 + BAP1 + promoters and enhancers. H , example loci.

Article Snippet: The sgRNA targeting BAP1 was obtained from Addgene (#125837).

Techniques: Binding Assay, Knock-Out, Infection, Plasmid Preparation, Expressing, Western Blot, ChIP-sequencing, Modification

Journal: The Journal of Biological Chemistry

Article Title: Colorectal cancer hot spot mutations attenuate the ASXL-MLL4 interaction

doi: 10.1016/j.jbc.2025.108333

Figure Lengend Snippet: Knockout of ASXL2 reduces MLL4 binding on MLL4 + BAP1 + enhancers. WT and ASXL2 KO HCT116 were subjected to Western blot and ChIP-seq analyses. A , Western blot analyses. B–D , ASXL2 KO reduces MLL4 binding on MLL4 + BAP1 + promoters and enhancers. Heat maps ( B ) and average profiles ( B ) were aligned around the center of MLL4 binding sites on MLL4 + BAP1 + promoters and enhancers. D , example loci.

Article Snippet: The sgRNA targeting BAP1 was obtained from Addgene (#125837).

Techniques: Knock-Out, Binding Assay, Western Blot, ChIP-sequencing

Journal: The Journal of Biological Chemistry

Article Title: Colorectal cancer hot spot mutations attenuate the ASXL-MLL4 interaction

doi: 10.1016/j.jbc.2025.108333

Figure Lengend Snippet: Genomic colocalization of BAP1 and MLL4 regulates expression of a subset of genes in HCT116. WT, BAP1 KO and MLL4 KO HCT116 were subjected to RNA-seq analyses. A , Pie chart showing gene expression changes in BAP1 KO and MLL4 KO cells. B , genes downregulated >2-fold in BAP1 KO or MLL4 KO cells. C , GO analysis of 78 genes decreased in both BAP1 KO and MLL4 KO. D , genes upregulated >2-fold in BAP1 KO or MLL4 KO cells. E , GO analysis of 136 genes increased in both BAP1 KO and MLL4 KO. F , FPKM values of BAP1 and MLL4 co-regulated genes. G , example loci of BAP1 and MLL4 co-regulated genes. H , Boxplot comparing the mRNA expression levels of MLL4, BAP1, and ASXL1 in normal colon tissues (GTEx, n = 308) versus colon (TCGA-COAD, n = 288). Expression values represent log2-transformed transcripts per million (TPM + 0.001) from RSEM. Statistical significance was assessed using the Mann-Whitney U test. Outliers (log2(TPM + 0.001) < −9) were excluded from visualization.

Article Snippet: The sgRNA targeting BAP1 was obtained from Addgene (#125837).

Techniques: Expressing, RNA Sequencing, Gene Expression, Transformation Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Colorectal cancer hot spot mutations attenuate the ASXL-MLL4 interaction

doi: 10.1016/j.jbc.2025.108333

Figure Lengend Snippet: CRC-relevant hot spot mutations in ASXLs. A and B , MST binding curves ( A ) used to determine binding affinities ( B ) of wild type and mutated MLL4 PHD2/3 for the indicated ASXL1, ASXL3 and ASXL2 peptides. The K d values represent average of four independent measurements, and errors represent standard deviation. n = 4 Point errors in ( A ) represent SEM. ( a ) taken from . C , alignment of amino acid sequences of ASXLs. CRC-relevant hot spot mutations are marked with red boxes. D and E , overlayed 1 H, N HSQC spectra of MLL4 PHD2/3 in the absence ( blac k) and presence of a 5-fold molar excess of indicated mutated ASXLs peptides. F , a model for the ASXL-dependent colocalization of the H3K4me1-specific methyltransferase MLL4 and the H2AK119ub-specific deubiquitinase BAP1 at enhancer and promoter regions of a subset of target genes.

Article Snippet: The sgRNA targeting BAP1 was obtained from Addgene (#125837).

Techniques: Binding Assay, Standard Deviation

Journal: Molecular Biology of the Cell

Article Title: Lysosome docking to WIPI1 rings and ER-connected phagophores occurs during DNAJB12- and GABARAP-dependent selective autophagy of misfolded P23H-rhodopsin

doi: 10.1091/mbc.E21-10-0505

Figure Lengend Snippet: Plasmids.

Article Snippet: lentiCRISPRv2 , Feng Zhang, Broad Institute , 52961.

Techniques: