Journal: FEBS letters

Article Title: Hepatocyte nuclear factor HNF1A is a potential regulator in shaping the super-enhancer landscape in colorectal cancer liver metastasis.

doi: 10.1002/1873-3468.14219

Figure Lengend Snippet: Fig. 2. Motif analysis for defined epicenters within SEs in CRC cell lines. (A) de novo enrichment analysis of TF binding motifs using ChIP- seq data obtained from public dataset (GSE77737). The top enriched motifs along with associated TFs are depicted and ranked by p values in representative cell lines. (B) The distribution of HNF1 motif instance near the H3K27ac peaks (‘epicenters’). The motif instances were recovered and mapped to SEs and distribution intensity was normalized to that of H3K27ac peak center in normal crypts. (C) Occupancy of master TFs within SEs and TEs. TFs binding events were counted according to putative binding sites within SEs and TEs with extended equivalent length. P value was adjusted using Bonferroni correction (**P < 0.01, ***P < 0.001, ****P < 0.0001). (D, E) Representative distri- bution of epicenters within a maintained (D) and a gained (E) SE. The putative binding sites of HNF1 family are indicated (arrows). The repre- sentative sequence of binding site is shown above. Black rectangular boxes indicate multiple H3K27ac peaks gained near binding sites.

Article Snippet: HNF1A antibody (89670) and ChIP sonication Chromatin IP kit (56383) were purchased from Cell Signaling Technology (Danvers, MA, USA). b-actin antibody (sc-47778) was purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Binding Assay, ChIP-sequencing, Sequencing

Journal: FEBS letters

Article Title: Hepatocyte nuclear factor HNF1A is a potential regulator in shaping the super-enhancer landscape in colorectal cancer liver metastasis.

doi: 10.1002/1873-3468.14219

Figure Lengend Snippet: Fig. 3. De novo motif analysis in isogenic CRC cell lines. (A) Identification of SEs in parental KM12C cells (Left) and matched KM12SM cells derived from xenografted liver metastasis (Right). Enhancers are plotted in an increasing order according to input-normalized signal. Enhancers above the inflection point of the curve are designated as the population of SE. SE-associated cancer-related genes are represented in red dot along with respective ranks and texts. (B) Tables depicting TF binding motifs enriched at defined epicenters within SEs in KM12C (Left) and KM12SM (Right). (C, D) Representative distribution of epicenters within HNF1 motif-enriched SE near ARHGAP26 and LRCH1 loci. The putative motif binding sites are indicated (arrows). The representative sequence of binding site is shown above. Black rectangular boxes indicate multiple H3K27ac peaks gained near TF binding sites.

Article Snippet: HNF1A antibody (89670) and ChIP sonication Chromatin IP kit (56383) were purchased from Cell Signaling Technology (Danvers, MA, USA). b-actin antibody (sc-47778) was purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Derivative Assay, Binding Assay, Sequencing

Journal: FEBS letters

Article Title: Hepatocyte nuclear factor HNF1A is a potential regulator in shaping the super-enhancer landscape in colorectal cancer liver metastasis.

doi: 10.1002/1873-3468.14219

Figure Lengend Snippet: Fig. 5. Downregulated genes are enriched in EMT-related pathways in HNF1A knocked-out KM12SM cells. (A) GREAT functional analysis of HNF1A motif-enriched SEs in KM12SM and parental cell line. The top 10 enriched Gene Ontology terms are shown. (B) Representative HNF1A motif-containing SEs in regulating Wnt signaling target gene CDH17. (C) Representative HNF1A motif-containing SEs in regulating Wnt signaling target gene S100A4. (D) RNA-Seq result of representative downregulated gene CDH17. (E) RNA-Seq result of representative downregulated gene S100A4. (F) RNA-Seq result showing downregulated genes are the most enriched in ECM organization signature. (G) Gene family categorizing of downregulated genes involved in ECM organization, Wnt, and TGF-b signaling.

Article Snippet: HNF1A antibody (89670) and ChIP sonication Chromatin IP kit (56383) were purchased from Cell Signaling Technology (Danvers, MA, USA). b-actin antibody (sc-47778) was purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Functional Assay, RNA Sequencing

Journal: FEBS letters

Article Title: Hepatocyte nuclear factor HNF1A is a potential regulator in shaping the super-enhancer landscape in colorectal cancer liver metastasis.

doi: 10.1002/1873-3468.14219

Figure Lengend Snippet: Fig. 6. HNF1A is upregulated in synchronous liver metastases. (A) Immunoblots of HNF1A in matched normal (N), primary tumor (P) and synchronous liver metastases (M) from CRC patients. The expression of b-actin served as the internal control. (B) Representative immunohistochemical staining of HNF1A in matched primary and metastatic samples. (C) The intensity of immunostaining was quantified using IMAGEJ (****P < 0.0001).

Article Snippet: HNF1A antibody (89670) and ChIP sonication Chromatin IP kit (56383) were purchased from Cell Signaling Technology (Danvers, MA, USA). b-actin antibody (sc-47778) was purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Staining, Immunostaining

Journal: Frontiers in endocrinology

Article Title: Expression of long noncoding RNA Xist is induced by glucocorticoids.

doi: 10.3389/fendo.2022.1005944

Figure Lengend Snippet: FIGURE 6 ChIP assay showing GR binding to Xist promoter region. (A) Schematic diagram of the approximate locations of GREs and flanking primers used in PCR reactions. (B–D) agarose gel images showing PCR products amplified from anti-GR antibody precipitated DNA fragments and primer pairs flanking the indicated GREs. Input: 2% sonicated DNA; H3: Anti-histone H3 mAb (positive control); IgG: normal rabbit IgG (negative control); GR: Anti-GR mAb. Experiment was performed 3 times with similar results. Shown is the results from one representative experiment.

Article Snippet: ChIP assays was performed using a SimpleChIP Plus Sonication Chromatin IP Kit (#56383) and a monoclonal antibody against GR (#3660S) according to the manufacturer’s instructions (Cell Signaling Technology, Inc.).

Techniques: Binding Assay, Agarose Gel Electrophoresis, Sonication, Positive Control, Negative Control

Journal: Molecular cell

Article Title: Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence

doi: 10.1016/j.molcel.2019.01.021

Figure Lengend Snippet: (A) Dot plot showing the enrichment of p300 and CBP over 55 Senescence-Activated SEs. (B) Dot plot showing the enrichment of p300 and CBP over op 55 Senescence-Activated TEs. (C-E) CBP and p300 occupancy at all Senescence-Deactivated SEs (C), top 1% Senescence-Deactivated TEs (D) and top 11 Senescence-Deactivated TEs (E). (F-I) Browser track views of p300/CBP signal at 4 SEs. (J) Western blot showing the shRNA-mediated depletion of p300 and CBP in cells used for RS assays in (K-N). (K-N) RLS curves of cells harboring shRNAs targeting CBP in multiple biological replicate experiments. (O) Representative viability plot during an RS assay with cells harboring CBP KD. (P) Plot showing p values of RLS experiments in (K-N) using a repeat measure analysis. The red line indicates the threshold for significance (p=0.05). (Q) Plot showing cumulative cell numbers in an oncogene-induced senescence assay with cells harboring p300, CBP or control hairpins. Asterisk indicates a p<0.0001 in a 2-way ANOVA test. (R) Representative viability plot during the oncogene-induced senescence assay shown in (Q). For p300/CBP ChIP-seq, proliferating cells were at PD 30 and 25, and senescent cells at pD 78 and 76.

Article Snippet: ChIP was performed as described previously from ~12e6 cells( Shah et al., 2013 ). p300 and CBP ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Western Blot, shRNA, Control, ChIP-sequencing

Journal: Molecular cell

Article Title: Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence

doi: 10.1016/j.molcel.2019.01.021

Figure Lengend Snippet: (A) Schematic showing samples for RNA- and PRO-seq libraries; (1) from proliferating and senescent uninfected cells and (2) senescent cells harboring control or p300-targeting hairpins. (B) Schematic showing the licensing of new enhancers in senescence with chromatin looping, engagement of transcription machineries and production of eRNAs and mRNAs. (C) Table showing the numbers of genomic elements (all, promoter, TE, SE and SE*) and their target genes used to draw box plots in D-G in proliferating and senescence conditions. SE* represents SEs with multiple acetylations. (D) Box plot of fold change of PRO-seq signal (senescence vs proliferating) across different genomic elements in senescence (TE, SE and SE*) measured at the enhancers (left) and the nearest target gene (right). (E) Same as (D) except the genomic elements are called in proliferating cells. (F) Box plot of fold change of RNA-seq signal (senescence vs proliferating or control vs KD) across different genomic elements called in senescence. (G) Box plot of fold change of RNA-seq signal (senescence vs proliferating) across different genomic elements called in proliferating cells. (H) Browser track views of PRO-seq signal at 3 SEs. 2-tailed Mann-Whitney-Wilcoxon Test was used to compare bins within and between boxplots. (I) ChIP signal of various histone acetylations at the promoters of the nearest genes identified as targets of the Senescence-Activated SEs. For PRO-seq, proliferating cells were at PD 20 and senescent cells at PD 76. For RNA-seq, proliferating cells were at PD 29 and 34 while senescent cells were at PD 78 and 79.

Article Snippet: ChIP was performed as described previously from ~12e6 cells( Shah et al., 2013 ). p300 and CBP ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Control, RNA Sequencing, MANN-WHITNEY

Journal: Molecular cell

Article Title: Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence

doi: 10.1016/j.molcel.2019.01.021

Figure Lengend Snippet: (A-C) RLS curves of cells harboring hairpins targeting p300 or a control. (D) qPCR showing KD efficiency of p300 hairpins relative to a housekeeping gene. (E) Representative viability plot during an RS assay with cells harboring p300 KD using trypan blue exclusion. (F) Plot showing p values of lifespan experiments in (A-C) and Figure S2A-X using a repeat measure analysis. The red line indicates the threshold for significance (p=0.05). (G) IF images showing TIF formation in cells harboring a control or p300 hairpin. TIFs (arrow) are assigned if there is co-localization of 53BP1 (green) and Cy3-labeled telomere end (red). Insets show a magnified view of the TIFs. (H) The number of TIFs per cell are counted and plotted. (I) Representative SA-β-gal staining in cells harboring control or p300 hairpins. (J) The number of β-gal positive cells represented as a percentage of total counted cells. (K) IF images showing EdU incorporation in cells harboring a control or p300 hairpin. (L) The percentage of EdU incorporation is estimated by counting the percentage of cells (DAPI, blue in K) that are stained with EdU (red in I) and plotted as a bar plot. (M) Western blots from 2 independent RLS experiments showing the downregulation of histone H3, lamin B1 and cyclin A1 during passage to senescence of cells harboring either p300 or control hairpin. Note that at comparable PDs (indicated in red), cells that are depleted in p300 retain more H3, lamin B1 and cyclin A1 compared to control. (N) Western blots showing protein levels of multiple HATs in proliferating and senescent cells including CBP and p300. n = number of cells scored, p value estimates are from unpaired t-tests.

Article Snippet: ChIP was performed as described previously from ~12e6 cells( Shah et al., 2013 ). p300 and CBP ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Control, Labeling, Staining, Western Blot