Journal: Frontiers in Endocrinology

Article Title: Liquid L-T4 therapy in hypothyroid patients with gastric diseases, an observational study

doi: 10.3389/fendo.2024.1386629

Figure Lengend Snippet: Prevalence of patients with TSH > 3.5 μIU/mL or TSH < 3.5 μIU/mL in the two groups of patients (T-LT4 or L-LT4 group) at each time-point of evaluation.

Article Snippet: The prevalence of patients with TSH<0.3 μIU/ml was 15.0% in the T-LT4 group against 16.9% in the L-LT4 group (chi-square, p=0.75).

Techniques:

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) Venn diagram of overlapping 5240 candidate CAD-associated variants (including those in high linkage disequilibrium at r 2 ≥0.8) with HCASMC ATAC-seq open chromatin regions ( n =323), H3K27ac ChIP-seq active enhancer regions ( n =462) or TF binding via TCF21 or AP-1 ChIP-seq ( n =193). Unique overlapping numbers shown for combined overlaps, respectively. ( b ) Forest plot depicting odds ratio (OR) of enrichment for CARDIoGRAMplusC4D (CAD), inflammatory bowel disease (IBD), ulcerative colitis (UC) or entire GWAS catalogue SNPs in individual or combined HCASMC data sets as calculated using the Fisher's exact test. Dots represent mean OR and lateral lines represent 95% confidence intervals. ( c ) Histogram distribution of globally normalized GWAS SNPs in regions centred on HCASMC open chromatin regions within a 1-kb window. ( d ) Heatmap distribution of HCASMC open chromatin regions centred on CTCF motif (from JASPAR) within a 0.5-kb window (left panel). Hierarchical clustering heatmap showing distribution of ATAC-seq open chromatin, JUN or TCF21 ChIP-seq binding regions centred on 5,240 CARDIoGRAMplusC4D SNPs (right panels). ( e ) Two-dimensional scatter plot of GWAS SNPs in HCASMC open chromatin regions showing most significant enrichment for cardiovascular (CARDIoGRAMplusC4D and coronary heart), brain and autoimmune phenotypes in upper right quadrant. Data shown are representative of n =10 biological replicates in HCASMCs cultured under normal growth conditions.

Article Snippet: Purified rabbit polyclonal antibody against H3K27ac (ab4729) was purchased from Abcam.

Techniques: ChIP-sequencing, Binding Assay, Cell Culture

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) LocusZoom plot showing results of CARDIoGRAMplusC4D 1000G fine-mapping at SMAD3 locus at chromosome 15q24.1 ( n =60,801 cases; n =123,504 controls) . Circles represent SNPs associated using an additive or recessive model, and colour-coded for LD ( r 2 ) with the lead SNP, rs56062135 (purple diamond), based on 1000G phase 1 v3 training data set. ( b ) LD plot generated from 1000G phase 1 augmented haplotypes in Europeans for SMAD3 locus, showing linked lead SNP, rs56062135, with candidate regulatory SNP, rs17293632. Colour-coded for LD based on D ′ values, shown as in boxes. ( c ) UCSC browser screenshot at SMAD3 locus, showing overlap of candidate SNP rs17293632 with ATAC-seq open chromatin tracks in coronary tissue ( n =3 biological replicates per condition) and HCASMCs treated under various conditions ( n =2 biological replicates per condition), TF-binding ChIP-seq tracks for TCF21, JUN and JUND, and active enhancer histone modification H3K27ac ChIP-seq ( n =4 biological replicates), as well as ENCODE layered H3K27ac for HUVEC (blue) and NHLF cells (purple). Inset, motifs in open chromatin regions showing alignment to reference sequence and position relative to rs17293632. Genomic coordinates refer to hg19 assembly. ( d ) Normalized ATAC-seq read counts for HCASMCs treated under various conditions and by genotype at rs17293632. Values represent mean±s.e.m. ( n =2 biological replicates for stimulations and n =5 biological replicates for different genotypes). ( e ) Allele-specific ChIP (haploChIP) for AP-1 proteins (JUN, JUNB and ATF3), TCF21 and H3K27ac in HCASMCs heterozygous at rs17293632. Values represent mean±s.e.m. of triplicates from a representative experiment ( n =5 biological replicates). * P <0.01 versus control, IgG or between two genotypes using an unpaired two-tailed t -test with Welch's correction for unequal variances.

Article Snippet: Purified rabbit polyclonal antibody against H3K27ac (ab4729) was purchased from Abcam.

Techniques: Generated, Binding Assay, ChIP-sequencing, Modification, Sequencing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

doi: 10.1038/ncomms12092

Figure Lengend Snippet: ( a ) UCSC browser screenshot at 9p21.3/ CDKN2B-AS locus, showing overlap of candidate SNP rs1537373 with ATAC-seq open chromatin tracks in coronary tissue ( n =3 biological replicates per condition) and HCASMCs treated under various conditions ( n =2 biological replicates per condition), TF-binding ChIP-seq tracks for TCF21, JUN and JUND, and active enhancer histone modification H3K27ac ChIP-seq ( n =4 biological replicates), as well as ENCODE layered H3K27ac for HUVEC (blue) and NHLF cells (purple). Inset, motifs generated from HOMER in open chromatin regions showing alignment to reference sequence and position relative to rs1537373 SNP. Genomic coordinates refer to hg19 assembly. ( b ) Normalized ATAC-seq read counts for HCASMCs treated under various conditions and by genotype at rs1537373. Values represent mean±s.e.m. ( n =2 biological replicates for stimulations and n =5 biological replicates for different genotypes). ( c ) Allele-specific ChIP (haploChIP) for AP-1 proteins (JUN, JUNB and ATF3), TCF21 and H3K27ac in HCASMCs heterozygous at rs1537373. Values represent mean±s.e.m. of triplicates from a representative experiment ( n =5 biological replicates). * P <0.01 versus control, IgG or between two genotypes using an unpaired two-tailed t -test with Welch's correction for unequal variances.

Article Snippet: Purified rabbit polyclonal antibody against H3K27ac (ab4729) was purchased from Abcam.

Techniques: Binding Assay, ChIP-sequencing, Modification, Generated, Sequencing, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: rs4903064 resides in a renal cancer–associated regulatory element. A , ATAC-seq tracks from primary renal cancer cells at the DPF3 locus. The top track indicates SNPs in high LD with rs4903064 (r 2 > 0.8). ATAC-seq was performed in cells from three individuals with different rs4903064 genotypes (TT, CT, CC). KIRC RE indicates r egulatory e lements defined by ATAC-seq in ccRCC TCGA samples. B , zoomed in view of ( A ). SNP rs4903064 resides in open chromatin and in KIRC_67261. Please note that open chromatin is only detected in cells carrying the risk allele C. C , sequences of the two alleles at rs4903064. The risk allele C creates a putative hypoxia-responsive element (HRE). D , allele-specific qPCR assay for rs4903064 indicates a shift toward the risk allele C in DNA fragments captured by ATAC experiments in tumor cells ( red triangle ) or tubular cells exposed to 1 mM DMOG ( orange triangle ) when compared to input DNA or DNA from ATAC experiments in untreated tubular cells. Cells were isolated from an individual heterozygous for rs4903064. E , allelic ratio of rs4903064 in ATAC samples from tubular cells isolated from three individuals heterozygous for the SNP performed in triplicates. Cells were exposed to 1 mM DMOG for 16 h or left untreated. Bars indicate mean values ± SD. t test, ∗∗∗∗ p < 0.0001. F , allelic ratio of rs4903064 in FAIRE samples from cells of four heterozygous individuals. Cells were exposed to 1 mM DMOG for 16 h, and values were normalized to corresponding input DNA. The mean value for each individual is shown. Bars indicate mean values ± SD. ∗one sample t test, p < 0.05. G , reporter assay in HeLa cells using the pGL3 promoter vector with or without a 415 bp sequence covering the protective or the risk allele of rs4903064. Cells were treated with 1 mM DMOG for 16 h as indicated. Bars indicate mean values ± SD of one experiment performed in triplicates. H , HeLa cells were depleted for the indicated HIF-α isoforms using siRNA and transfected with the reporter plasmid including the risk allele. Cells were exposed to 1 mM DMOG for 16 h. Reporter activity was normalized to the activity of co-transfected β-galactosidase. Bars indicate mean values ± SD of one experiment performed in triplicates. I and J , DPF3 protein levels are high in HIF-positive ccRCC tumors. A total of 330 ccRCC tumors were stratified according to the immunoreactive score for HIF-1α, HIF-2α, and DPF3 staining in no, low, medium, or strong staining. Fractions of DPF3 signals are shown for each subgroup of HIF staining. Pearson χ 2 -test; p = 1.8 × 10 −8 for HIF-1α ( I ) and p = 0.003 for HIF-2α ( J ). ATAC, Assay for Transposase-Accessible Chromatin; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; FAIRE, formaldehyde-assisted isolation of regulatory elements; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Isolation, Reporter Assay, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: HIF binding to the RCC risk allele. A , HIF ChIP qPCR results for the DPF3 locus. Experiments were performed from cells isolated from 11 TT, 11 CT, and 1 CC individuals. Bars indicate mean values ± SD. Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01. B , binding of HIF to the control locus at EGLN3 is not affected by the rs4903064 genotype. Bars indicate mean values ± SD. C , HIF ChIP-seq tracks in two different ccRCC cell lines at the rs4903064-associated enhancer (TT: RCC4 and CC: individual 40,911,432 from Yao et al. ). HIF binding is only detectable in cells from a patient carrying the C allele ( red , HIF-1α: 12 × C from 12 reads in total, HIF-2α 99 × C from 99 reads in total). D , same tracks as in ( C ) covering the EGLN3 control locus. E and F , H3K27ac ChIP-seq tracks from corresponding normal ( blue ) and ccRCC ( red ) tissue indicates increased activity of the disease-associated regulatory element at DPF3 ( E , highlighted in yellow ) and of the EGLN3 locus ( F ) in tumors. G , H3K27ac ChIP-seq tracks for two different ccRCC cell lines at the rs4903064-associated enhancer (TT: 786-O and CC: individual 40,911,432 from Yao et al. ). Levels of the active enhancer mark H3K27ac are high in the individual carrying the C allele and reduced upon VHL re-expression. H , same tracks as in ( G ) covering the EGLN3 control locus. Note that H3K27ac levels are reduced upon VHL re-expression in both cell lines. ccRCC, clear cell renal cell carcinoma; ChIP, chromatin immunoprecipitation; DPF3, double PHD fingers 3; EGLN3, Egl-9 family hypoxia inducible factor 3; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism; VHL, von Hippel-Lindau.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Binding Assay, ChIP-qPCR, Isolation, Control, ChIP-sequencing, Activity Assay, Expressing, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Allele specific interactions at rs4903064. A – C , allele-specific qPCR on ChIP samples generated from primary tubular cells exposed to 1 mM DMOG for 16 h. A , HIF ChIP indicates enrichment for the C allele in DNA fragments captured with HIF-1α or HIF-1β antibodies. n = 7 individuals. B , H3k27ac as a marker for active chromatin is enriched at the C allele. n = 4 individuals. C , RNApol2 is enriched at the C allele of rs4903064 indicating increased interaction with the transcriptional machinery from this allele. n = 4 individuals. D , quantification of the results in ( A – C ). The allelic ratio is shifted to the risk allele C, which indicated increased interaction with HIF and the transcriptional machinery as well as increased activity of this allele. Values are mean ± SD. One sample t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ChIP, chromatin immunoprecipitation; DMOG, dimethyloxalylglycine; HIF, hypoxia-inducible transcription factor.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Generated, Marker, Activity Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

doi: 10.1016/j.jbc.2022.101699

Figure Lengend Snippet: Regulation of DPF3 expression and effect of DPF3 on tubular cell growth. A , relative expression levels of DPF3 in primary renal tubular cells exposed to 1 mM of DMOG for 16 h. Samples were stratified according to the rs4903064 genotype (TT = 42, CT/CC = 44). Values were normalized to expression levels of the housekeeping gene HPRT and to values from corresponding untreated cells. Each dot represents the mean value from cells of one individual. qPCR was performed in duplicates per individual. Bars indicate mean values ± SD. Student’s t test, ∗∗∗∗ p < 0.0001. B , DPF3 expression levels in isolated tumor cells compared to corresponding untreated tubular cells from 8 (TT) and 14 (CT/CC) individuals, respectively. Bars indicate mean values ± SD. One sample t test, ∗ p < 0.05. C , top , allelic ratios of rs4903064 in cells from heterozygous individuals exposed to DMOG 1 mM or left untreated. One sample t test, ∗∗∗ p < 0.001. Bottom , allelic ratios of rs4903064 in ccRCC cells with high (CA9+) or low (CA9−) CA9 expression as a marker for HIF-1α activity. Values are mean from technical triplicates, and each dot represents values derived from one individual. Bars indicate mean values ± SD. t test, ∗ p < 0.05. D , Western blot for DPF3 and beta actin from lysates of different clones of HKC-8 cells with or without defective DPF3. E , Western blot for DPF3 and beta actin from lysates of two different huPTC lines transfected either with nontargeting (nt) guides or guides targeting DPF3. F , proliferation assays of the clones of cells indicated in ( D ). Dark lines and dots indicate the mean value from the three different clones of cells, which were tested in biological triplicates. Light gray and light blue lines indicate individual values for the three different clones of cells. G , proliferation assay from cells indicated in ( E ). H , Western blot for DPF3 and beta actin from lysates of primary renal tubular cells (PTC) transfected either with nontargeting (nt) guides or guides targeting DPF3. Cells were harvested at day 3 after transfection. I , cyst volume of PTC 7 days after plating in a Matrigel matrix (n = 3 wells per condition, 4 quadrants per well). J , representative images of the cysts in the Matrigel matrix. Values are mean ± SD. t test, ∗ p < 0.05, ∗∗ p < 0.01 for F , G , and I . CA9, carbonic anhydrase 9; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; huPTC, human urinary primary tubular cells.

Article Snippet: ChIP experiments were performed as previously described using antibodies directed against HIF-1α (Cay10006421, rabbit polyclonal, Cayman Chemicals), HIF-1β (NB100–110, rabbit polyclonal, Novus Biologicals), RNApol 2 (SC-899, rabbit polyclonal, Santa Cruz), or H3K27ac (ab4729, rabbit polyclonal, Abcam) ( , ).

Techniques: Expressing, Isolation, Marker, Activity Assay, Derivative Assay, Western Blot, Clone Assay, Transfection, Proliferation 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: 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