Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: Heatmap shows the distribution of p300, H3K27ac and H3K27me3 signals around +/-5kb region of p300 only peak and p300/H3K27ac common peaks. B. Pie chart shows the distributions of annotated features around p300 only peaks in MEL cell. C. Homer motif analysis identifies significant enrichment of transcription factors binding sites at p300 only peaks. D. Binding profiles of transcription factors at p300 only peak in MEL cells. E. Distribution of different types of interaction observed at TAD boundary(blue) and chromatin loops(red). X axis represents number of different types of interactions. “Gene” represents genes that do not overlap with H3K27me3 and p300 only peak. “Intergenic” represents intergenic regions that do not overlap with H3K27me3 and p300 only peak. F and G. Aggregated peak analysis (APA) of chromatin loops anchored by p300 only peaks (F) and p300 only peak-H3K27me3 peaks (G) in G1ER late G1 cell. HiC matrix was normalized with VC_SQRT, window = 6. H. ECDF plot shows the comparison of the expression level of H3K27me3 non-overlapping and overlapping genes looped to p300 only peaks at TAD boundary or Chromatin loops. MEL cell RNA-seq processed expression quantification data was obtained from ENCODE. Wilcoxon test was used to compare the log2(TPM+1) value of H3K27me3 genes and non-H3K27me3 genes, P values ≤ 0.05 indicating a significant difference.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: Binding Assay, Comparison, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: A. Location of primers used for ChIP-qPCR in C and D. B. Cpox intron 5 TFBS KO gel image. C. Cpox intron 5 TFBS KO BLAST result, MEL p300 ChIP seq data from ENCODE. D. Sanger sequencing data, black line shows the deletion site.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: ChIP-qPCR, ChIP-sequencing, Sequencing

Journal: bioRxiv

Article Title: Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction

doi: 10.1101/2023.12.05.570115

Figure Lengend Snippet: UCSC genome browser tracks shows p300, H3K27ac, H3K27me3, and H3K4me1 pattern at Dcbld2 promoter and Cpox intron 5. ChIP-seq data from ENCODE. p300 only peak was highlighted in grey. B. qRT-PCR result shows the expression of Dcbld2-2 , St3gal6-2 and CpoxeRNA after Cpox intron 5 TFBS deletion in UMEL cells. Three biological replicates. Data are mean ± s.d., unpaired one-tailed t-test. C. ChIP-qPCR result shows p300 binding at the Cpox intron 5 region after Cpox knock down by shRNA in UMEL cells. Two biological replicates. Data are mean ± s.d., unpaired one-tailed t-test. D. Model of Cpox mRNA loss activate neighboring gene and enhancer. Left graph shows in normal situation, right graph shows Cpox mRNA knock down situation. Colored arrows represent the genomic locus of the protein genes and enhancer. Colored curves represent the corresponding RNA transcribed. Blue dots represent p300, orange dots represent PRC2. Rectangles on genes represent the promoters.

Article Snippet: For p300 ChIP experiment, undifferentiated MEL cells were used, p300 antibody (Santa Cruz Biotechnology, sc-48343).

Techniques: ChIP-sequencing, Quantitative RT-PCR, Expressing, One-tailed Test, ChIP-qPCR, Binding Assay, Knockdown, shRNA

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: The PHGDH ACT domain is required for forming PHGDH/p300/cMyc/AF9 axis and regulates cMyc transactivation. A) Schematic diagram of human PHGDH protein and its five domains. aa, amino acid. B) HA‐tagged cMyc and FLAG‐tagged PHGDH (including WT and five domain truncates) were transiently transfected into HEK293T cells. Co‐IP was performed with an antibody against FLAG. Antibodies against HA and FLAG were used to detect the association between cMyc and PHGDH. HA was used as an input control. dSB1, SB1 domain depletion; dNB, NB domain depletion; dSB2, SB2 domain depletion; dASB, ASB domain depletion; dACT, ACT domain depletion. C) GST‐tagged ACT domain and His‐tagged cMyc protein were purified from Escherichia coli , and a GST pull‐down assay was performed by incubating both the recombinant proteins together. GST was used as a blank control. Antibodies against His and GST were used to detect the association between cMyc and the ACT domain. D) cMyc transcriptional activity was measured by the Dual‐Luciferase Reporter Assay System according to the manual using PHGDH ‐depleted PLC/PRF/5 and Hep3B cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Cells expressing shNT were used as control. rPHGDH‐dACT, shRNA‐resistant PHGDH‐dACT (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). E) Chromatin immunoprecipitation (ChIP) analysis of cMyc enrichment on promoters of five canonical cMyc targets was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t ‐test, n = 3). F) qRT‐PCR assay of the five genes from (E) (mean ± SD, two‐tailed Student's t ‐test, n = 3). G) Co‐IP analysis of p300, cMyc, and PHGDH was performed using PHGDH ‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Antibody against p300 was used to enrich p300 associated complex. Immunoblotting analysis of PHGDH, cMyc, and p300 was performed using the indicated antibodies. H) p300 was transiently depleted by specific siRNA in PHGDH ‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Immunoblotting analysis of cMyc‐AcK148, cMyc, p300, and PHGDH was performed using the indicated antibodies. NC, negative control. I) Co‐IP analysis of cMyc, p300, and PHGDH was performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Antibody against cMyc was used to enrich the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies.

Article Snippet: A histone H3Kac antibody (Clone 2G1F9, 1:200 for ChIP, a POL2 antibody (Clone 4H8, 1:100 for ChIP), and a p300 antibody (Clone NM11, 1:200 for ChIP, 1:1000 for IB, 1:100 for IP) were purchased from Active Motif.

Techniques: Transfection, Co-Immunoprecipitation Assay, Control, Purification, Pull Down Assay, Recombinant, Activity Assay, Luciferase, Reporter Assay, Expressing, shRNA, Chromatin Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: PHGDH/cMyc axis drives CXCL1/IL8 expression. A–D) RNA‐sequencing analyses were performed using PHGDH‐depleted PLC/PRF/5 cells rescued with rPHGDH‐WT or rPHGDH‐dACT. Gene Ontology (GO) enrichment analyses of the differentially expressed genes were presented (A). GSEA enrichment plot of the KEGG pathway NOD‐like receptor‐related gene was shown in (B). The correlation of all NOD‐like receptor‐related gene expression with the phgdh expression status was displayed by the ranking metric score. A positive score indicates a correlation with the rPHGDH‐dACT and a negative score indicates a correlation with rPHGDH‐WT; The red indicates a gene that contributes most to the enrichment result and the blue indicates a gene that contributes less (C). The total differentially expressed genes (FC>2 or FC<0.5; p value <0.05) were displayed using a volcano plot. FC, fold change of rPHGDH‐dACT compared to rPHGDH‐WT (D). E) In PHGDH ‐depleted PLC/PRF/5 cells with rPHGDH‐WT or rPHGDH‐dACT or further depletion of c‐Myc, qRT‐PCR validated the top up‐regulated genes from (C) (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). F) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PHGDH ‐depleted PLC/PRF/5 cells, which were rescued with rPHGDH‐WT or rPHGDH‐dACT (mean ± SD, two‐tailed Student's t ‐test, n = 3). G) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. H) Co‐IP analysis of cMyc, p300, and PHGDH was performed using PLC/PRF/5 cells expressing WT or K148R mutant Myc. Antibody against cMyc was used to enrich the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies. I) qRT‐PCR validated CXCL1/IL8 and IL1B genes using PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). J) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium culturing PLC/PRF/5 cells expressing WT or K148R mutant Myc (mean ± SD, two‐tailed Student's t‐test, n = 3). K) Immunoblotting analysis of CXCL1/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. L) ChIP analysis of PHGDH, cMyc, p300, RNA Pol II (Pol 2), AF9, and H3Kac on CXCL1 gene promoter was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t ‐test, n = 3).

Article Snippet: A histone H3Kac antibody (Clone 2G1F9, 1:200 for ChIP, a POL2 antibody (Clone 4H8, 1:100 for ChIP), and a p300 antibody (Clone NM11, 1:200 for ChIP, 1:1000 for IB, 1:100 for IP) were purchased from Active Motif.

Techniques: Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Two Tailed Test, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Control

Journal: Cell Stem Cell

Article Title: Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

doi: 10.1016/j.stem.2020.09.001

Figure Lengend Snippet: PRS Locus EC1.45 and EC1.25 Are Active in hCNCCs and during Mouse Craniofacial Development (A) ChIP-seq and ATAC-seq for PRS locus putative enhancer clusters EC1.45 (p300 Peak1 and Peak2), EC1.35 (S1 and S2), and EC1.25 (S3–S6). (B) Luciferase reporter assays for EC1.45, EC1.35 (S1 and S2) and EC1.25 (S3–S6) in hCNCCs (left) and hESCs (right). (C) Schematic outlining craniofacial domains at E9.5 and E11.5. BA1-2, branchial arch 1-2; FNP, frontonasal prominence; LNP, lateral nasal process; MdP, mandibular process; MNP, medial nasal process; MxP, maxillary process. (D) In situ hybridization (ISH) for Sox9 at E9.5 and E11.5. (E) Mouse LacZ reporter assay for EC1.45, EC1.35 (S1 and S2), and EC1.25 (S3–S6 tested individually) at E9.5 and E11.5. (F) HREM for an EC1.45 LacZ reporter embryo at E11.5 (frontal view, top; parasagittal section, bottom). White arrow, activity in the MdP. See also Figure S3 and and .

Article Snippet: Rabbit polyclonal p300 (discontinued) – ChIP , Santa Cruz Biotechnology , Cat# sc-585; RRID: AB_2231120.

Techniques: ChIP-sequencing, Luciferase, In Situ Hybridization, Reporter Assay, Activity Assay

Journal: Cell Stem Cell

Article Title: Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

doi: 10.1016/j.stem.2020.09.001

Figure Lengend Snippet: Dissection of EC1.45 Enhancer Region Uncovers a Core Role of the Coordinator Motif and TWIST1 Binding in Developmental Enhancer Regulation (A) TWIST1 ChIP-seq peaks (marked under track) at EC1.45 overlap p300 Peak1 and Peak2 and minimally active sequences (min1 and min2). (B) Luciferase assay for EC1.45 min1 and min2, tested separately and combined, along with Coordinator mutant sequences. Left: schematic of the constructs. (C) Coordinator motif (top; Prescott et al., 2015 ) compared with the motif enriched at TWIST1 binding sites in hCNCCs (bottom). (D) Luciferase assay for the heterologous enhancer sequence for human min1 plus vertebrate min2. Left: schematic of the constructs. A scatterplot depicts the luciferase signal compared with the sum of Coordinator scores (ANOVA p = 0.00035; right). (E) TWIST1 is upregulated during hCNCC differentiation and reduced in chondrocytes (fragments per million [FPM]). (F) Schematic of plasmids, primers, and probes for ChIP-ddPCR for wild-type (WT) and Coordinator mutant (4x mut) min1+min2 plasmids. F, forward; R, reverse. (G) TWIST1 ChIP-ddPCR for P4 late hCNCCs transfected with the plasmids in (F), normalized to input, and WT adjusted to 1. Two biological replicates are depicted. See also Figure S5 .

Article Snippet: Rabbit polyclonal p300 (discontinued) – ChIP , Santa Cruz Biotechnology , Cat# sc-585; RRID: AB_2231120.

Techniques: Dissection, Binding Assay, ChIP-sequencing, Luciferase, Mutagenesis, Construct, Sequencing, Transfection

Journal: Cell Stem Cell

Article Title: Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder

doi: 10.1016/j.stem.2020.09.001

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal p300 (discontinued) – ChIP , Santa Cruz Biotechnology , Cat# sc-585; RRID: AB_2231120.

Techniques: Recombinant, Membrane, Clinical Proteomics, Purification, Derivative Assay, Knock-Out, Protease Inhibitor, DNA Extraction, Virus, Hybridization, Library Quantification, Multiplex Assay, Immunoprecipitation, Reporter Assay, Capture-C, Sequencing, CRISPR, Expressing, Plasmid Preparation, In Situ, Software, Imaging, Microscopy