Journal: Nucleic Acids Research

Article Title: Transcriptional induction by ecdysone in Drosophila salivary glands involves an increase in chromatin accessibility and acetylation

doi: 10.1093/nar/gkaf284

Figure Lengend Snippet: E23 expression in the salivary glands suppresses ‘active’ ESEs co-bound with EcR and CBP/Nejire at 20E-activated loci. ( A ) Venn diagram reflecting an intersection of EcR and CBP/Nejire peaks located in 20E-activated loci ±5 kb from a total number of EcR and CBP/Nejire peaks in these loci defined by ChIP-Seqs in salivary glands of hsp-e23 wandering larvae in untreated conditions. Regions co-bound with CBP/Nejire and EcR were termed ESEs. ( B ) A scheme displaying two principles of classification of ESEs. Location: ESEs located in ±250 bp of TSSs were defined as proximal ESEs, ESEs located outside of the TSSs but within gene loci, ±5 kb were called distal ESEs. Activity: ESEs are categorized based on their levels of H3K27Ac acetylation. Those with high levels of H3K27Ac are considered ‘active’. ESEs with lower levels of H3K27Ac are ‘poised’. Hierarchical clustering was used to separate ESEs into groups based on their activity levels (provided in ). Created in BioRender ( https://BioRender.com/0ngjjab ). ( C ) Average distribution of EcR, CBP, FAIRE, H3K27Ac enrichment estimated by ChIP-Seqs at ‘active’ proximal (located ±250 bp of TSSs, N = 64) and active distal (located outside of the TSSs but within gene loci, ±5 kb, N = 258) ESEs in 20E-activated primary loci in salivary glands in control condition (NHS) and after treatment of hsp-e23 larvae 20–22 h before pupariation with double 1-h heat shock (with a 1-h rest at RT) (HS). ( D ) Average distribution of EcR, CBP, FAIRE, H3K27Ac proteins estimated by ChIP-Seqs at ‘poised’ proximal (located ±250 bp of TSSs, N = 135) and ‘poised’ distal (located outside of the TSSs but within gene loci, ±5 kb, N = 369) ESEs in 20E-activated primary loci in salivary glands in control condition (NHS) and after treatment of hsp-e23 larvae 20–22 h before pupariation with double 1-h heat shock (with a 1-h rest at RT) (HS). For panels (C) and (D) ChIP-Seq binding levels were calculated as a ratio to Input (for FAIRE and H3K27Ac the Input was subtracted from sample). The X-axis represents the distance to the ESE in kbp. Average profiles were calculated as a median of binding level with the standard error displayed on the profiles. The FC was calculated using normalized coverage within 500 bp around the summit peak for EcR, CBP, FAIRE and within 1000 bp around the summit peak for H3K27Ac of the analysed ESEs (as a ratio of NHS signal to HS signal). The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( E ) Scheme displaying that the transcriptional response to 20E in salivary glands involves activating a subset of ESEs with high H3K27Ac levels, highlighting the importance of 20E in regulating chromatin state and EcR binding dynamics. Created in BioRender ( https://BioRender.com/n650dzg ).

Article Snippet: Antibodies against histone H3K27Ac (39133) were purchased in Active motif.

Techniques: Expressing, Activity Assay, Control, ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Transcriptional induction by ecdysone in Drosophila salivary glands involves an increase in chromatin accessibility and acetylation

doi: 10.1093/nar/gkaf284

Figure Lengend Snippet: ‘Active’ ESEs, sensitive to 20E depletion, are tissue-specific, as evidenced by our and previously published data. Average distribution of EcR, CBP, FAIRE and H3K27Ac enrichment estimated by ChIP-Seqs at ( A ) ‘active’ proximal (located ±250 bp of TSSs, N = 64) ESEs and ( B ) ‘active’ distal (located outside of the TSSs but within gene loci, ±5 kb, N = 258) ESEs in salivary glands (SG) and brain (BR) of hsp-e23 wandering larva in untreated conditions. ChIP-Seq binding level was calculated as a ratio to Input. For FAIRE-Seq and H3K27Ac the Input was subtracted from sample. The X-axis represents the distance to the ESE in kbp. Average profiles were calculated as a median of binding level with the standard error displayed on the profiles. The FC was calculated using normalized coverage within 500 bp around the summit peak for EcR, CBP, FAIRE and within 1000 bp around the summit peak for H3K27Ac of the analysed ESEs (as a ratio of SG signal to a BR signal). The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( C ) Average FAIRE-Seq signals from the previously published data (from ) at distal (N = 258) and proximal (N = 64) ‘active’ ESEs of 20E-activated targets in salivary glands. The FC was calculated using normalized coverage within 500 bp around the summit peak of the analysed ESEs (as a ratio of SG signal to Wing and Leg signals). The FC for proximal ‘active’ loci were 5.1 for SG/Wing and 4.0 for SG/Leg. The FC for distal ‘active’ loci were 9.7 for SG/Wing and 13.2 for SG/Leg. The results of the paired t -test analysis are provided on the graphs, where ‘**’ means P ≤ .01. ( D ) PCA plot represents a strong correlation between the FAIRE-Seq signals for the salivary glands obtained previously and in a current study. In other tissues the chromatin accessibility levels at the described ESEs varied. For the PCA analysis FAIRE-Seq signals were calculated using normalized coverage within 500 bp around the summit peak of the analysed ESEs. ( E ) Model illustrating the regulation of 20E-activated target loci in salivary glands. We suggest that 20E-activated targets are regulated by a tissue-specific set of ‘active’ ESEs. In salivary glands, these ‘active’ ESEs have CBP/Nejire and EcR peaks, as well as increased chromatin accessibility and acetylation, whereas in the brain, they are silent. Created in BioRender ( https://BioRender.com/okxy4sl ).

Article Snippet: Antibodies against histone H3K27Ac (39133) were purchased in Active motif.

Techniques: ChIP-sequencing, Binding Assay

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Effects of oxygen tension on retinal levels of HIF-1α, VEGF and β3-AR from PD7 to PD17. ( A ) Schematic diagram of the OIR model including DMOG administration daily from PD7 to PD12. ( B ) Representative blots showing protein levels of HIF-1α, VEGF and β3-AR as evaluated by Western blot in retinal extracts at different times from normoxic controls or OIR mice without or with DMOG administration. β-actin was used as the loading control. ( C – E ), Relative densitometric analyses of the protein levels of HIF-1α, VEGF and β3-AR. ( F ) Retinal mRNA levels of β3-AR at different times from controls or OIR mice untreated or treated with DMOG. * p < 0.05 vs. normoxic controls. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Western Blot, Control, Comparison

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Schematic representation of mouse and human β3-AR genes including their upstream sequences. ( A ) In the mouse gene, 5 exons (E1–E5; solid boxes) and 4 introns (dashed lines) are depicted. They potentially express up to 6 different alternative mRNAs of which 3 codify for the canonical β3-AR protein (yellow mRNAs) while the other 3 for an alternative β3-AR protein with a different C-terminal sequence (purple mRNAs). The putative transcription-start site (TSS) is indicated by the red arrow. The positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal HBS consensus sequence (underlined sequence 5′-ACGTG-3′). ( B ) In the human gene, the positions of the 6 potential HBSs relative to the TSS are in green. All of them contain the minimal consensus sequence (underlined sequence 5′-ACGT-3′). The putative TSS is indicated by the red arrow. The highly conserved nucleotides G −2 and/or C +5 in the mouse and human HBSs sequence are highlighted in red.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Sequencing

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α modeling and HIF-1/DNA docking. ( A ) Root-mean-square (RMS) displacement of protein backbone (black arrow indicates the time at which the stabilization of the protein structure occurs). ( B ) RMS fluctuation of aminoacid displacement relative to the starting structure and the principal domains of the HIF-1α protein, accordingly colored in ( C ). ( D ) HIF-1α protein modelized in its dimeric form showing the correct interaction with the DNA fragment. The two monomers are reported in green and orange respectively, while the DNA fragment is highlighted in blue. The binding site generated by dimerization is better shown in the focus section.

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Binding Assay, Generated

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: Docking analysis of model 1 and model 3. ( A ) HIF-1/HBS #1 best association complex: full structure and focus on HIF-1-DNA interactions (boxes). ( B ) HIF-1/HBS #3 best association complex: full structure and focus on HIF-1-DNA interactions (boxes).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques:

Journal: Cells

Article Title: HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina

doi: 10.3390/cells11081271

Figure Lengend Snippet: HIF-1α interaction with HBS #1 and corresponding β3-AR gene expression at PD12 (from 0 to 12 h of hypoxia) or at PD17. ( A ) Schematic diagram of the OIR model pointing to the specific times under analysis. ( B ) Data from HIF-1α chromatin immunoprecipitation and HBS #1-specific qPCR (ChIP-qPCR) represented as fold enrichment relative to IgG input. ( C ) Corresponding levels of β3-AR mRNA. White bars represent data from retinas of normoxic controls while grey bars represent data from hypoxic mice. One-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Each histogram represents the mean ± SEM of data from 6 independent samples. * p < 0.05 vs. normoxic controls ( n = 6 samples).

Article Snippet: Blots were blocked in 3% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal against HIF-1α (ab2185; Abcam, Cambridge, UK; 1:500 dilution), rabbit polyclonal against VEGF (ab9570; Abcam; 1:1000 dilution), mouse monoclonal against β3-AR (sc-515763; Santa Cruz Biotechnologies, Santa Cruz, CA, USA; 1:200 dilution), mouse monoclonal against β-actin (A2228; Sigma Aldrich, St. Louis, MO, USA; 1:2500 dilution).

Techniques: Gene Expression, Chromatin Immunoprecipitation, ChIP-qPCR, Comparison

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Summary of genome-wide STAT5 binding sites at L1. (A) The Venn diagram shows the number of identified STAT5A and STAT5B sites (peaks) in AABB tissue and STAT5B sites in BB tissue. (B) Average peak heights of STAT5A and STAT5B in AABB and BB tissues were estimated after library size normalization (RPM, reads per 10 million, input subtracted). (C) Mean fold changes of STAT5A and STAT5B target genes in AABB tissue and STAT5B target genes in BB tissue were calculated. The genes containing STAT5 peaks within ±1 kb around TSSs were regarded as STAT5 target genes. (D) Normalized tag counts (RPM) of STAT5A, RNA polII and H3K4me3 from 200 bp around STAT5A peak centers at L1 were calculated and compared between AABB and BB . Log 2 -transformed values were used ( x and y axes). (E) Normalized tags of H3K4me3 at positions 1 kb upstream and 2 kb downstream of TSS were summed up and divided by the size (3 kb) and then quantile normalized for comparison (top). The scatter plot shows the fold change ( x -axis) and difference ( y -axis) of H3K4me3 average enrichment between genes (spot) in AABB and BB . Cutoffs for significant changes were set as follows: 1.5-fold change ( x -axis, AABB/BB ) and four average tag difference ( y -axis, AABB/BB ). Among the genes showing significant changes of H3K4me3 enrichment, the number of STAT5 target and non-target genes was counted (bottom). (F) Genome browser views represent three gene loci ( Wap , Csn1s2a and Stap1 ) showing changes of H3K4me3 level and one housekeeping gene locus ( Actb ).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Genome Wide, Binding Assay, Transformation Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages in early pregnancy. ( A ) Transplanted tissues from mice expressing Stat5a or Stat5b at various levels as indicated were harvested on day 6 of pregnancy and stained with H&E. At this stage alveolar development in all samples is sparse in all epithelial cells expressing Stat5 and is even more reduced in Null cells (f). Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Staining of the membrane transporter molecule NKCC1, which is downregulated as epithelial cells differentiate, indicates a more mature developmental stage in wild type (a) cells, intermediate maturity in cells with two or one Stat5 alleles (b and c) and strong staining in Null cells (f). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Expressing, Membrane

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages at parturition. The nomenclature of mice with the different genotypes is based on the alleles they have retained. We refer to wild-type mice and Stat5ab fl/fl mice as AABB mice; Stat5ab fl/fl;MMTV- Cre (with Stat5ab -deficient mammary epithelial cells) as Null mice; Stat5a −/− mice as BB mice; Stat5b −/− mice as AA mice; Stat5ab +/ null mice as AB mice. Mice carrying only a single functional allele of either Stat5a ( Stat5ab null /Stat5b − ) or Stat5b ( Stat5ab null /Stat5a − ) are referred to as A mice and B mice, respectively. ( A ) Transplanted mammary tissues obtained from mice of different genotypes were collected on the day of parturition and analyzed by histology. Alveoli are expanded and filled with milk in the presence of four (a) and two (b and c) Stat5 alleles. Epithelial cells with only one active Stat5 allele (d and e) form dense alveoli lacking signs of secretory activity. Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Mammary tissues of transplanted epithelia obtained from mice of different genotypes were collected at parturition and sections were stained with anti-NKCC1 antibody (red) and α-smooth muscle actin (green). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Functional Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 regulation of and binding to known target genes

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of the casein gene cluster. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means higher conservation) . Expression level of five milk protein genes was measured by both RNA-seq and qRT-PCR (bottom left). Absolute expression level of the milk protein genes is shown (bottom right).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of STAT5 target genes. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means more conserved). ( A ) Wap gene, ( B ) Cish gene, ( C ) Socs2 gene, ( D ) Bcl6 gene and ( E ) Stat5a/b genes.

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: (A) Schematic of generating human embryonic stem cell (hESC) models that harbor heterozygous growth syndrome-associated mutations in DNMT3A and NSD1 by CRISPR-Cas9 genome engineering. Schematic was created using BioRender. See for detailed genotypes of the mutant clones. (B) Relative expression of DNMT3A transcripts normalized to RNA18S transcript levels. Each dot represents an independent clone. (C) Western blot analysis of DNMT3A protein expression. DNMT3A knockout (KO) clones were included as controls. HDAC1 was used as a loading control. Each lane represents an independent clone. DNMT3A genotypes are as follows: WT, WT/WT; frameshift, WT/frameshift; R882H, WT/R882H; KO, frameshift/frameshift; GoF, WT/W330R or WT/D333N. The plots on the right show the relative intensity of DNMT3A bands, normalized to HDAC1. (D) Relative expression of NSD1 transcripts normalized to RNA18S transcript levels. (E) Mass spectrometry of histones H3.1 and H3.3 K36 modifications in WT and NSD1 LoF hESCs. Each dot represents an independent clone. unmod., unmodified; me1, monomethylated; me2, dimethylated; me3, trimethylated; ac, acetylated. For panels B, C, D, and E, Statistical significance was determined by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques: CRISPR, Mutagenesis, Clone Assay, Expressing, Western Blot, Knock-Out, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: Proportions of in each full-stack chromatin state for (A) DNMT3A LoF hESCs, (B) TBRS patient blood (HypoMPs n=832), (C) DNMT3A GoF hESCs, (D) a HESJAS patient peripheral blood leukocyte sample (HypoMPs n=2,796; HyperMPs n=9,576), (E) NSD1 LoF hESCs, and (F) Sotos syndrome patient blood (HypoMPs n=24,148; HyperMPs n=4,168) Background represents proportion of all probes in each state. HyperMPs of TBRS patients were not analyzed due to a small size (n=38). Statistical significance was determined by Fisher test. *, p<0.001.

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

doi: 10.1101/2025.07.08.663614

Figure Lengend Snippet: (A) Number of overlapping HypoMPs between DNMT3A LoF and GoF mutants. P<2.2*10^-16, Fisher’s exact test of unique DNMT3A GoF in all probes compared to DNMT3A GoF shared with DNMT3A LoF. (B) Log2 odds ratios showing enrichment of shared HypoMPs from DNMT3A mutants across full-stack chromatin states. Top 10 enriched states are shown (all p<1*10-18). (C) Mean DNA methylation values at CpG positions within selected enhancer chromatin states. Each dot represents an independent clone. Statistical significance was determined by Student’s t-test. *, p<0.05; **, p<0.01; ***,p<0.001; ns, not significant. (D-E) RELI analysis of shared HypoMPs intersected with >10,000 public chromatin datasets. Shown are the top 200 datasets ranked by Z-score. Most enriched chromatin factors (D) and cell types (E) are highlighted. PSC TF: POU5F1, NANOG, SOX2, cohesion: RAD21, NIPBL, PRC1.1: BCOR, KDM2B, PCGF1, RYBP, RNF2. Controls represent randomly sampled EPIC probe regions. Each dot represents a dataset profiling a chromatin factor in a human cell type. Supplemental Table 2 contains RELI results of all examined datasets. PSC, pluripotent stem cells; TF, transcription factors. (F) CUT&RUN (H2AK119ub) or ChIP-seq (all others) signal in WT hESCs 10 kilobases upstream and downstream from the center of shared HypoMPs or matched control regions. BCOR, KDM2B, PCGF1, H3K36me2 occupancy data are from GEO accession number GSE104690, RNF2 data is from GSE105028, H3K36me3 is from ENCODE (ENCSR476KTK), and H2AK119ub is from GSE301386 (this study).

Article Snippet: Membranes were incubated overnight at 4 °C with a primary antibody against DNMT3A (C-12, Santa Cruz Biotechnology, sc-365769), followed by HRP-conjugated anti-mouse IgG secondary antibody (Cell Signaling Technology, #7076) for 1 hour at room temperature.

Techniques: DNA Methylation Assay, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Non-canonical enhancers control gene expression and cell fate in human pluripotent stem cells

doi: 10.1101/2025.06.01.657118

Figure Lengend Snippet: (A) Quantitative RT-PCR for indicated genes before (Control) or after DOX-induced expression of dCas9-KRAB (CRISPRi) and gRNA-mediated targeting to primed enhancers in hiPSCs. 2 -ΔΔCt ± s.d.; normalisation using GAPDH (housekeeping gene) and uninduced hiPSCs as a reference (= 1). (B) Quantitative RT-PCR for indicated genes before (Control) or after DOX-induced expression of dCas9-VP16 (CRISPRa) and gRNA-mediated targeting to primed enhancers in hiPSCs. 2 -ΔΔCt ± s.d.; normalisation using GAPDH (housekeeping gene) and uninduced hiPSCs as a reference (= 1). (C) Live-cell imaging of hiPSCs without (Control) or with (CRISPRi) the addition of DOX and TMP to induce the expression of dCas9-KRAB, marked by GFP expression (green). Constitutive expression of three gRNAs targeting the primed enhancer of SLC13A4 . (D) Proportion of remaining cells (in %) before (Day 0) or after addition of DOX and TMP (Day1-4) for dCas9-KRAB expression during live-cell imaging. Three (3 gRNAs) or individual gRNAs (gRNA1-3) for targeting the enhancer of APLN or SLC13A4 were constitutively expressed. (E) Immunofluorescence images of hiPSC colonies before (Control) or after addition of DOX and TMP (Day 1-4) for dCas9-KRAB expression, marked by GFP expression. Three gRNAs targeting the enhancer of S LC13A4 were expressed constitutively. Samples were stained for DNA, SOX2, and NANOG. (F) Quantification of fluorescence intensity of SOX2 and NANOG in images shown in (E). (G) Heatmap shows relative expression levels of genes +/- 1Mb of the SLC13A4 locus based on RNA-seq data for hiPSCs after the addition of DOX and TMP for 1 day (D1) or 2 days (D2) to induce dCas9-KRAB with constitutive expression of gRNAs targeting the enhancer of SLC13A4 in comparison to control hiPSCs. (H) Volcano plot shows differentially expressed genes of RNA-seq data for hiPSCs after the addition of DOX and TMP for 1 day to induce dCas9-KRAB with constitutive expression of gRNAs targeting the enhancer of SLC13A4 in comparison to control hiPSCs. (I) Coverage tracks of STARR-seq, ATAC-seq, ChIP-seq (H3K27ac, H3K4me1) showing the SLC13A4 enhancer region in hiPSCs, and H3K9me3 and CTCF CUT&Tag tracks showing the same SLC13A4 enhancer genomic region in control and enhancer CRISPRi conditions. Chr = chromosome.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies against SOX2 (Santa Cruz, sc-365823, 1:500) and NANOG (R&D -AF1997, 1:100) in PB.

Techniques: Quantitative RT-PCR, Control, Expressing, Live Cell Imaging, Immunofluorescence, Staining, Fluorescence, RNA Sequencing, Comparison, ChIP-sequencing

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: cAMP signaling induces CRTC2 nuclear translocation and condensate formation. a) Live‐cell imaging of ectopically expressed CRTC2‐eGFP, CREB‐eGFP, CREM‐eGFP, and ATF1‐eGFP in 293T cells without (DMSO) and with forskolin (FSK) treatment (upper). Quantification of condensates number per nucleus (lower). b) In vitro droplet formation assay with recombinant CRTC2‐IDR‐eGFP at different protein concentrations (left). Quantification of the size of droplets (right). c) Representative images of the in vitro FRAP experiment with recombinant CRTC2‐IDR‐eGFP (upper). Quantification of FRAP data for CRTC2‐IDR‐eGFP puncta (lower). d) In vitro droplet formation assay of recombinant eGFP fusion proteins fused with wild‐type (WT) CRTC2‐IDR or CRTC2‐IDR mutants (left). Quantification of the size of droplets (right). e) Live‐cell imaging of ectopically expressed CRTC2‐eGFP in 293T cells. Arrows indicate representative CRTC2 puncta that fused over time. The dotted line area indicates the nucleus. f) Representative images of the FRAP experiment with ectopically expressed CRTC2‐eGFP in 293T cells (upper). The dotted line area indicates the nucleus. Quantification of FRAP data for CRTC2‐eGFP puncta (lower). g) Live‐cell images of ectopically expressed WT CRTC2‐eGFP or CRTC2‐IDR‐R>A mutant (R>A‐eGFP) in 293T cells (left). Quantification of cells with eGFP foci and western blot analysis of CRTC2‐eGFP or CRTC2‐IDR‐R>A expression (right). h) Live‐cell snapshots of ectopically expressed mCherry‐CRY2 fusion proteins fused with WT CRTC2‐IDR (upper) or CRTC2‐IDR‐R>A mutant (lower) in 293T cells before and after blue light stimulation (left). Quantification of cells with mCherry foci before and after blue light stimulation (right). Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. n.s., not significant. Scale bar, 5 µm (a), 10 µm (b,d). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Translocation Assay, Live Cell Imaging, In Vitro, Tube Formation Assay, Recombinant, Mutagenesis, Western Blot, Expressing

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: P‐TEFb interacts with CRTC2 and enhances CRTC2‐dependent transcription. a) Immunofluorescence analysis of the co‐localization of CRTC2 and CycT1 in HeLa cells treated with DMSO or FSK (left). Line scans along the dotted lines in the images (right). b) Co‐IP assay examining the interaction between endogenous CRTC2 and P‐TEFb in 293T cells treated with DMSO or FSK for 1 h. c) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells treated with the indicated concentration of FSK or H89. d) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells ectopically expressing Myc‐SIK1. e) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 (WT or S171/274A mutants) in 293T cells. f) Quantification of CRE‐luc luciferase activity in 293T cells transfected with the indicated amounts of FLAG‐CDK9 plasmids and treated with DMSO or FSK (10 µ m ) for 6 h. g) Quantification of CRE‐luc luciferase activity in 293T cells treated with flavopiridol (FP, 300 n m ) and FSK (10 µ m ) for 6 h. h) Quantification of CRE‐luc luciferase activity in HA‐CRTC2‐expressing 293T cells infected with lentivirus carrying sgRNA against CDK9 (sgCDK9) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. i) Quantification of CRE‐luc luciferase activity in FLAG‐CDK9‐expressing 293T cells infected with lentivirus carrying sgRNA against CRTC2 (sgCRTC2) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001; n.s., not significant. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Concentration Assay, Expressing, Luciferase, Activity Assay, Transfection, Infection

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 extracts P‐TEFb from HEXIM1‐containing inhibitory complex. a) Live‐cell imaging of 293T cells co‐expressing CDK9‐mCherry and CRTC2‐eGFP or R>A‐eGFP and treated with DMSO or FSK (10 µ m ) for 1 h (left). The dotted line area indicates the nucleus. Quantification of cells with CDK9‐mCherry foci upon treatment with DMSO or FSK (right). b) Immunofluorescence analysis of co‐condensates of CRTC2‐IDR‐mCherry‐CRY2 with CDK9 or CycT1 in 293T cells before or after blue light stimulation (left). Quantification of cells with CRTC2‐IDR co‐condensates with CDK9 or CycT1 (right). c) Live‐cell imaging of 293T cells co‐expressing CycT1‐CFP, HEXIM1‐RFP, CRTC2‐eGFP, and R>A‐eGFP as indicated. d) Quantification of cells containing CycT1‐CFP foci. e) RNA‐IP assay examining the association between FLAG‐CDK9 and 7SK snRNA in 293T cells co‐expressing HA‐CRTC2 or HA‐R>A and treated with DMSO or FSK. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Live Cell Imaging, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 forms nuclear droplets in cystic epithelial cells. a) Western blot analysis of CRTC2 levels in kidneys from WT and Pkd1 −/− mice ( n = 3). b) Representative immunohistochemistry (IHC) images of CRTC2 in kidneys of WT and Pkd1 −/‐ mice (left). Quantification of the signal intensity of nuclear CRTC2 (right) ( n = 5). Cy, large cyst. c) Representative IF images of CRTC2 in the kidney of WT and Pkd1 −/‐ mice. Cy, large cyst. d) Representative IHC images of CRTC2 in kidneys from healthy people ( n = 5) and ADPKD patients ( n = 19). e) Quantification of the signal density of nuclear CRTC2 of (d). f) Correlation between the signal intensity of nuclear CRTC2 and eGFR in patients with ADPKD. Pearson's correlation coefficients are displayed for each graph. p values for (f) were determined using linear regression analysis. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. *** p < 0.001, * p < 0.05. Scale bar, 20 µm (b–d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Western Blot, Immunohistochemistry

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Loss of CRTC2 delays cyst formation in an ADPKD mouse model. a) Experimental design of the ADPKD mouse model. b) Representative kidneys from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). c) KW/BW ratios of the indicated groups of mice in the ADPKD model. The number below each bar refers to the number of samples analyzed. d) Hematoxylin and eosin (H&E) staining of kidney sections from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). e) Cystic index of H&E‐stained kidneys from mice at P29 with indicated genotype. The number below each bar refers to the number of samples analyzed. f) Plasma BUN levels of mice from the indicated groups. The number below each bar refers to the number of samples analyzed. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. Scale bar, 2 mm (b,d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Staining, Clinical Proteomics

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Genome‐wide CRTC2 localization in human ADPKD cells. a) Representative immunofluorescence images of endogenous CRTC2 in WT 9–12 cells. The result is from more than three independent experiments. b) Western blot analysis of whole‐cell lysates from WT 9–12 cells with indicated treatment. The result is from more than three independent experiments. c) Genomic distribution of CRTC2 in WT 9–12 cells. d) Heatmaps of normalized ChIP‐seq signals for CRTC2, H3K4me1, and H3K4me3, H3K27ac. The rows show 3 kb flanking the CRTC2 peak center. e) Boxplots of the normalized counts of H3K4me1, H3K4me3, and H3K27ac signals at CRTC2 binding + or − peaks. f) Boxplots of CRTC2, H3K4me1, H3K4me3, and H3K27ac reads in the indicated groups. Low: CRTC2 reads < 39; medium: CRTC2 reads 40–70; high: CRTC2 reads > 71. g) ChIP‐seq tracks of CRTC2, H3K4me3, H3K27ac, and H3K4me1 on representative genes. h) Motif analysis of CRTC2 peaks. i) GO and KEGG pathway enrichment analyses of CRTC2‐binding genes. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001, ** p < 0.01. Scale bar, 20 µm (a).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Genome Wide, Immunofluorescence, Western Blot, ChIP-sequencing, Binding Assay

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 regulates the expression of cystogenesis‐associated genes in human ADPKD cells. a) Heatmap of gene expression in WT 9–12 cells transfected with sgControl or sgCRTC2. Rows show the Z scores calculated for each group. b) Volcano plots showing differentially expressed genes. c) Gene set enrichment analysis displaying CRTC2‐binding gene set enriched for genes downregulated in sgCRTC2 cells. NES, normalized enrichment score. d) Venn diagram showing the overlap of sgCRTC2 downregulated genes and CRTC2‐binding genes, referred to as CRTC2‐target genes. e) GO and KEGG pathway enrichment analyses of CRTC2‐target genes. f) Heatmap of expression values of genes enriched in the indicated processes. Rows show the Z scores calculated for each group. g) RT‐qPCR analysis of mRNA levels of representative CRTC2‐target genes in kidneys from the indicated mouse groups. Data are presented as means ± SEM. The unpaired Student's two‐sided t ‐test was used for statistical analysis. **** p < 0.0001. These results are from three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Expressing, Gene Expression, Transfection, Binding Assay, Quantitative RT-PCR

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 activates cystogenesis‐associated genes by promoting paused Pol II release. a) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in the kidneys of WT and Pkd1 −/‐ mice ( n = 3). b) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in WT 9–12 cells (left). Line scan of the dashed line in the merged inset image (right). c) Western blot analysis of CRTC2 expression in WT 9–12 cells transfected with the indicated sgRNAs and constructs. d) Immunofluorescence analysis of ectopically expressed TY1‐CRTC2 and TY1‐CRTC2‐R>A in WT 9–12 cells with endogenous CRTC2 knocked out. e) RT‐qPCR analysis of representative CRTC2‐target genes in WT 9–12 cells transfected with the indicated sgRNAs and constructs. f) Cumulative curve of Pol II pausing index (PI) for CRTC2 target genes in normal and ADPKD kidney tissues. g) ChIP‐qPCR analysis of CycT1, Pol II Ser2P, and Pol II occupancy on LDHA and PLAU genes in WT 9–12 cells with endogenous CRTC2 replaced by TY1‐CRTC2 or TY1‐CRTC2‐R>A mutant. The inset bar graphs indicate the Pol II pausing index for LDHA and PLAU genes, respectively. h) Schematic illustration of how CRTC2 triggers the productive elongation of cystogenesis‐associated genes, ultimately leading to ADPKD progression. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 20 µm (a,b). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Western Blot, Expressing, Transfection, Construct, Quantitative RT-PCR, ChIP-qPCR, Mutagenesis

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Top: Venn diagram depicting differentially expressed genes (nascent RNA-seq, log2 fold-change >2 and FDR <0.05) in Thap1−/− versus WT and Thap1−/−Brca1Δ11 versus Brca1Δ11 MEFs in relation to THAP1-bound genes (ChIP-seq). The number of genes that were shown to be bound by THAP1 and were either downregulated or upregulated in THAP1-deficient MEFs are shown in blue and red, respectively.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: RNA Sequencing, ChIP-sequencing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Western blot analysis of doxycycline-dependent expression of exogenous SHLD1 (left) and THAP1 (right) proteins in WT MEFs 24 to 96 hours after induction with doxycycline (Dox) as detected by anti-Flag antibody.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Western Blot, Expressing

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A-B) Quantification of RPA2 (A) and RAD51 (B) foci in individual EdU-positive (S-phase) nuclei of WT, Brca1Δ11, Trp53bp1−/−Brca1Δ11 and two individual clones of Thap1−/− Brca1Δ11 MEFs. Cells were irradiated with 10 Gy and analyzed 4 h post-IR. Statistical significance was determined by Welch’s t-test.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Clone Assay, Irradiation

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: (A) Representative flow cytometry plots of IgM-to-IgA class switch recombination (CSR) in WT, Trp53bp1−/−, Shld1−/−, Shld3−/− and two individual clones of Thap1−/− (#4 and #12) CH12-F3 cells 24 hours after cytokine stimulation (IL-4, CD40L and TGFβ). Unstimulated WT cells are shown as a negative control. Quantification of IgM-to-IgA CSR is shown on the right and represents mean ± s.d., n=3.

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Flow Cytometry, Clone Assay, Negative Control

Journal: Molecular cell

Article Title: The Dystonia Gene THAP1 Controls DNA Double Strand Break Repair Choice

doi: 10.1016/j.molcel.2021.03.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ChIP-seq was performed as described previously ( Shinoda et al., 2019 ) with a rabbit polyclonal antibody against THAP1 (Proteintech, 12584–1-AP).

Techniques: Purification, Blocking Assay, Virus, Bacteria, Expressing, CRISPR, Knock-Out, Recombinant, Transfection, Cloning, PCR Cloning, Protease Inhibitor, Ligation, Library Quantification, Selection, Flow Cytometry, Cell Viability Assay, cDNA Synthesis, SYBR Green Assay, Cell Culture, Mutagenesis, Illumina Sequencing, Software, Microscopy, Imaging, Irradiation

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Translocation of CD44 from cytoplasm to nuclear in the reprogramming process of C3A cells . A. Real-time PCR analysis of CD44 in indicated time points of reprogramming process. Relative gene expression of CD44 to C3A cells was calculated for C3A-D5, C3A-D15, C3A-D25 C3A-D35, C3A-iCSCs P5 and C3A-iCSCs P45. Data are presented as the means ± SD from three independent. B. Immunofluorescence staining of CD44 in indicated time points of reprogramming process. Scale bar, 25μm. C. Western blot analysis of total protein (left) and cytoplasmic/nuclear protein (right) of CD44 in C3A and C3A-iCSCs. D. Immunohistochemical staining of CD44 in clinical liver cancer samples. Arrows indicated nuclear CD44-positive staining. Scale bar, 30μm.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Translocation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: CD44 transport was mediated by importin β and affected by importin α. A. The interaction between CD44 and importin β / transportin 1 was demonstrated by co-immunoprecipitation assay in C3A-iCSCs. B. Western blot analysis of flag after CD44-NLS-del / CD44-WT was transduced into C3A-iCSCs. C. Immunofluorescence staining analysis of flag after CD44-NLS-del / CD44-WT was transduced into C3A-iCSCs. Scale bar, 25μm. D. Immunofluorescence staining analysis of CD44 (red) and importin β (green) after Importazole was treated in C3A-iCSCs. DMSO indicated negative control, WGA indicated positive control. Scale bar, 10μm. E. Immunofluorescence staining analysis of CD44 (red) and importin β (green) after importin α was knocked down by RNA interfere assay. Scale bar, 10μm. C-E. Percentages of colocalization in ROI were qualified.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Negative Control, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Nuclear CD44 participated in naïve genes transcriptional regulation. A. Real-time PCR analysis of the naïve pluripotent genes KLF2 , KLF5, ZFP42, ESRRB , DNMT3L, GBX2, DPPA4 and LIFR in C3A cells and C3A-iCSCs. B. Real-time PCR analysis of CD44 and naive pluripotent genes after CD44 was silenced. C. ChIP-qPCR performed using CD44-specific antibodies in C3A cells and C3A-iCSCs. KLF2 , KLF5 , ESRRB represented their promoters respectively. Samples were analyzed by real-time PCR. Error bars showed the standard deviation of three independent ChIP-qPCR assays. D. Luciferase activity assay performed in C3A-iCSCs. Luciferase reporter plasmid pGL3-KLF2 / pGL3-KLF5 / pGL3-ESRRB containing their own promoter sequences was co-transfected with Renilla control vector and Ctrl / si-CD44 RNA sequences in C3A-iCSCs. Luciferase activities were measured after 72 hrs and presented as relative to the activity of Renilla luciferase. (* p <0.05, ** p <0.01, *** p <0.001, ns indicated no significant differences)

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Real-time Polymerase Chain Reaction, ChIP-qPCR, Standard Deviation, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Proposed model of CD44 transport from cytoplasm to nuclear . CD44 transport was mediated by importin β. Nuclear CD44 participated in transcriptional regulation of KLF2 , KLF5 , and ESRRB genes in C3A-iCSCs.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques:

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Representative RFX family transcription factors are grouped according to functional domains. The first group consists of RFX1, RFX2, and RFX3, which have all the domains, including AD (activation domain), DBD (DNA binding domain), B (domain B), C (domain C), and DIM (dimerization domain). The second group consists of RFX4, RFX6, and RFX8, harboring the domains of DBD, B, C and DIM domains. The third group consists of RFX5 and RFX7, which only have the DBD. The green and red squares mark the start and stop codon positions, respectively. (B) Western blot with indicated antibodies in the whole cochlea at different developmental stages. Different developmental stages include E13.5, E15.5, E17.5, P0, P7, and adult (2M). (C,D,E) Single cell-level expression patterns of the RFX family TFs in three developmental stages in the mouse cochlea. IPhC: inner phalangeal cells/border cells, OC/OSC: Claudius cells/outer sulcus cells, L.PsC lateral prosensory cells, M.PsC medial prosensory cells, Oc90: Oc90+ cells, IPhc/IBC, inner phalangeal cell /inner border cell; ISC/OSC/CC: Claudius cells and inner and outer sulcus cells.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Functional Assay, Activation Assay, Binding Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A-C) Immunofluorescence detection of Rfx3 protein in organ of Corti at different developmental stages. All protein immunostaining signals are in red, nuclei were counterstained with DAPI (blue). Hereafter is the same. (A) Rfx3 expression on E17.5 cochlea section. (B) Rfx3 expression on P7 cochlea section. The white dashed boxed region was magnified and displayed below. Hereafter is the same. (C) Rfx3 expression on adult cochlea section. (D-F) Immunofluorescence detection of Rfx7 proteins in organ of Corti at different stages. (D) Rfx7 expression on E17.5 cochlea section. (E) Rfx7 expression on P7 cochlea section. (F) Rfx7 expression on adult cochlea section. (G-I) RNA in situ detection of Rfx3 gene in organ of Corti at different developmental stages. All the RNA expression signals were labeled in green. The cochlea sections from E17.5 (G), P7 (H), and adult (I) wild-type stages revealed a dynamic expression pattern of Rfx3 gene during the development. (J-L) RNA i n situ detection of Rfx7 gene on the cochlea sections. Rfx7 showed dynamic expression patterns from E17.5 (J), P7 (K), and adult stage (L). The white triangles indicate the position of hair cells. Representative images from n>3 experiments. Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Immunofluorescence, Immunostaining, Expressing, In Situ, RNA Expression, Labeling

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Distance of Rfx3 ChIP-seq peaks to TSS positions. (B) Genomic distribution of Rfx3-enriched regions. (C) Localization of the Rfx3-motif within the peak sequence revealed a typical binding feature of transcriptional factor. (D) The most enriched gene ontology (GO) terms of the Rfx3-bound genes. (E) The top 5 most enriched motifs in the Rfx3-bound CREs, were revealed by the Homer Known motif analysis. (F) Rfx3 gene locus view revealed its regulatory features, including deposition of active marker H3K27ac, chromatin accessibility (ATAC-seq), and self-regulation by Rfx3. Two Rfx3-bound regions were indicated by red dashed box at the TSS and intron regions of Rfx3 gene locus. (G) Rfx7 gene locus view revealed its regulatory features, including H3K27ac deposition, chromatin accessibility, Rfx3 and Six1 binding.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Marker

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) The Venn diagram of 1543 common peaks between the E17.5 Rfx3 bound cis-regulatory elements (CREs) at E17.5 and E16.5 H3K27ac enriched regions. (B) There are 2979 common peaks between Rfx3 and hair cell specific ATAC-seq peaks. (C) Venn diagram showed 2501 common peaks between Rfx3 and spiral ganglion neuron (SGN) specific ATAC-seq peaks. (D) Comparisons between E17.5 Rfx3 ChIP-seq, P1 hair cell ATAC-seq, and P7 SGN ATAC-seq peaks together. Clustered heatmaps of Rfx3, H3K27ac, Hair cell ATAC-seq and SGN ATAC-seq peaks respectively within a -3 kb/+ 3 kb window. (E) Strong enrichments of Rfx3, Six1, and ATAC-seq signals in the promoter of Foxj1 genes. (F) Strong binding of Rfx3, however, no obvious deposits of H3K27ac, Six1 and Rfx3 in Dnah5 gene locus. (G) The large majority of 1543 common peaks (A) between Rfx3 and H3k27ac locate in or around the promoter region. (H) The Venn diagram shows that most of 2955 intergenic Rfx3 bound CREs do not have H3K27ac signals.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Single-cell level profiling of hair bundle related gene expresses in the adult cochlea. (B) RNA in situ detections of Triobp (upper panels) and Myo1h (lower panels) genes on cochlear sections in organ of Corti at P8. The Triobp and Myo1h probes were labelled in green, and nuclei were counterstained with DAPI in blue. The triangles indicate hair cells). (C) Genome browser visualization of Rfx3 peaks at the intron of Triobp gene. The RFX and SIX motifs were seen around the peak center. The red dashed box indicates Rfx3 bound regions. (D) Genome browser visualization of Rfx3 peaks at the TSS and intronic regions of Myo1h (E) Transient (G0) transgenic analysis of a 965-bp Rfx3-bound CRE (the red-dashed-boxed-region in ) in Triobp gene at P0 stage. This +40 kb intronic CRE ( Triobp +40000) drives specific expression of GFP reporter in hair cells (in 4/4 transgenic lines). Top panels, images of whole cochlea; middle panels, higher magnification of the partial areas in the top panel; bottom panels, images of cochlear sections showing GFP+ hair cells in the organ of Corti. (F) Transient (G0) transgenic analysis of an 847-bp Rfx3-bound CRE in the Myo1h intronic region (the red-dashed-boxed-region in , right) at P0 stage. The +70 kb intronic CRE ( Myo1h +70000) did not show expression activity in P0 cochlea (5/5, n=5 transgenic lines). Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: In Situ, Transgenic Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) The Venn diagram shows that there are 740 common CREs bound by Rfx3 (E17.5) and Six1 (E16.5). (B) SIX motif was one of the top 5 most enriched motifs in the 740 co-bound CREs, as revealed by the Homer Known motif analysis. (C) The Venn diagram shows 138 common peaks between Rfx3 and Foxp1 binding CREs in neural stem cells (NSC). (D) Homer motif analysis of 138 co-bound peaks identifies RFX as one of the most enriched motifs in Foxp1-bound CREs. (E) Single-cell level profiling of the Forkhead family TFs gene expressions in the adult cochlea. (F) The whole amount and section of immunofluorescence showing the expression pattern of Foxp1 in organ of Corti (P7). Top panels, whole cochlea duct. Bottom panels, images of cochlear sections. (G) Vangl2+22000 enhancer contains RFX, SIX and FOX-motifs. (H) Genome browser visualization of Rfx3 peaks at the TSS and intron of Vangl2 , as indicated by the red dashed boxes The tested CRE region is highly conserved as revealed by the conservation score track (black, bottom). (I) Genome browser visualization of Rfx3 peaks at the TSS and intron of Celsr1. Three Rfx3-bound regions were indicated by the red dashed boxes. Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Binding Assay, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Expression of RFX TFs, and hair cell differentiation and maintenance genes at E16, P7 and adult stages in inner ears. (B) Genome browser visualization of Rfx3 peaks at the Ikzf2 locus. Two Rfx3-bound regions were seen in the upstream intergenic and 3’ TTS regions. Left panels, higher magnification of the areas indicated by dashed lines at TSS; middle panels, whole Ikzf2 locus view; right panels, higher magnification of the upstream intergenic region. The Rfx3 bound CREs were indicated by the red dashed box3s. (C) Genome browser visualization of Rfx3 peaks at the CREs of Insm1 and intron of Tbx2 . (D) The schematic figure describing the dynamic expression pattern of Rfx3 and Rfx7 during inner ear development (left, E17.5; middle, P7; right, adult). The arrows indicate a shift from the transcriptional regulatory role towards a possible immune response function.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Expressing, Cell Differentiation