Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Impact of Eβ on topology, structural protein deposition, and transcription of Vβ segments. (A and B) Schematics and histograms of 3C data for the Trbv5 (A) and Trbv23 (B) viewpoints (anchors) in RAG-deficient DN thymocytes or pro-B cells (see for details). (C) Published ChIP-seq profile for CTCF in RAG-deficient DN thymocytes (top; ). (C and D) ChIP-qPCR for CTCF (C) and RAD21 (D) binding at the indicated sites in WT or mEβ thymocytes versus RAG-deficient pro-B cells. Data are presented as mean values for percent input signal from at least three independent experiments (±SEM). (E) Germline transcription of Trbv segments as monitored by RT-qPCR assays in the indicated cell types. Mean values from three independent experiments after normalization to signals for Actb are shown (±SEM). Thymocytes were pooled from 5–10 mice per experiment. Significant differences between WT and mEβ samples are denoted as *, P < 0.05 (Student’s t test).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-sequencing, ChIP-qPCR, Binding Assay, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See for details of cartoon data summaries. Here, red shading indicates that Trbv -Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-qPCR, Binding Assay, Quantitative RT-PCR, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

doi: 10.1084/jem.20141479

Figure Lengend Snippet: Identification of a Trbv tethering point in the RC flank. (A–E) 3C data for Trbv5 (A; the bottom shows ChIP-seq track for CTCF in DN thymocytes as well as locations of repetitive elements), 5′PC (B; schematic shown on top for 5′PC viewpoint; see ), Trbv3 (C), Trbv12-2 (D), and Trbv23 (E) viewpoints (anchors) in RAG-deficient DN thymocytes (WT, ΔPDβ1, or ΔminPDβ1 mice) or pro-B cells (see for details). (F) ChIP-qPCR for CTCF and RAD21 at 5′PC in the indicated cell types. All data are represented as means (±SEM) of three independent experiments. Thymocytes were pooled from 5–10 mice for each 3C or ChIP assay. Significant differences are denoted as *, P ≤ 0.05 (Student’s t test between WT and ΔPDβ1 genotypes).

Article Snippet: The following antibodies were used: CTCF (Rockland), Rad21 (Abcam), H3ac (EMD Millipore), H3K4me2 (Abcam), H3K4me3 (Abcam), H3K9me2 (Abcam), H3K27me3 (Abcam), and IgG (Santa Cruz Biotechnology, Inc.).

Techniques: ChIP-sequencing, ChIP-qPCR

Journal: Developmental biology

Article Title: Trim33 is required for appropriate development of pre-cardiogenic mesoderm

doi: 10.1016/j.ydbio.2019.03.018

Figure Lengend Snippet: A, heat map showing Trim33 ChIP-Seq read density around the merged peak regions. B, signal intensity plot representing Trim33 and input ChIP-Seq profiles around the merged peak regions. C, pie chart displaying distributions of Trim33 peaks across promoter, intragenic and intergenic regions. D, functional annotation analysis using GREAT reveals that Trim33 preferentially binds to genes associated with stem cell maintenance and mesoderm formation (GO: Biological Process), and holoprosencephaly, VSD and craniosynostosis (GO: Disease Ontology (developmental diseases)). E, three most common consensus motifs recognized by Trim33 are Ctcf, Tead and Isl1 motifs. F, venn diagram showing the overlap of Trim33, Ctcf and H3K27ac target genes as identified by ChIP-Seq. G, examples of genome browser images depicting different Trim33, Ctcf and H3K27ac profiles. H, ChIP-qPCR at enriched regions found near the four indicated genes; IP with the anti-Trim33 antibodies (controls, closed circles; mutants, open circles; n = 5 for Cbfa2t2l; n = 3 for Ctgf, Gse1 and Phc2; *,p < 0.05). I, co-immunoprecipitation of Trim33 with Ctcf and Smad2 in EBs transfected with Flag-tagged Trim33 cDNA and un-tagged Ctcf cDNAs with or without Activin stimulation (100 ng/ml, 40 min); n = 3.

Article Snippet: Transfection, immunoprecipitation and western blot assays EBs at day 6 of differentiation were dissociated with Trypsin, plated and transfected with full-length Myc/Flag-tagged mTrim33 cDNA (Origene MR227454) and full-length mCtcf cDNA (Origene MC202667) using ViaFect Transfection reagent (Promega PRE4981).

Techniques: ChIP-sequencing, Functional Assay, ChIP-qPCR, Immunoprecipitation, Transfection

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 1. ZFP143 can be homozygously tagged and stably binds to chromatin (A) Overview of genome-engineered cell lines. (B) Depletion western blots. On the left, ZFP143 (top) or CTCF (bottom) time courses for clones A and B. On the right, clones D and 3041 at the 3 h time point. TATA- binding protein (TBP) was used as a loading control. Black borders indicate cropping of whitespace or superfluous lanes. (C) Live-cell imaging of a clone A mESC colony showing nuclear localization of both CTCF and ZFP143. (D) Growth curve showing live-cell counts in clone D with (blue) and without (gray) constitutive ZFP143 depletion for 6 days. On day 3, cells were reseeded at a density of 250,000 live cells per well. Error bars indicate 95% confidence interval (CI) (n = 6). (E) Example FRAP images in the H2B-Halo, Halo-CTCF, and ZFP143-Halo conditions at 10 s before bleaching, on the bleach frame, 20 s after, and 600 s after bleaching. (F) FRAP curves showing normalized intensity of ZFP143 recovery (left) for clones A (magenta) and B (blue) and CTCF recovery (right) for clones A (magenta), B (blue), and D (orange) as well as the C87 Halo-CTCF line14 (red). Halo-NLS and H2B-Halo controls shown in gray. Error bars indicate 95% CI. (G) Example images of particles from SPT. (H) Jump displacement histograms for CTCF (clone D, left) and ZFP143 (clones A and B, right) for seven time lags. (I) Cumulative distribution functions (CDFs) comparing jump lengths between clones A and B (left) and clone D and C87 (right). (J) Summary of key measurements ±1 SD from SPT, FRAP, and absolute abundance quantification. See also Figure S1.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: Stable Transfection, Western Blot, Clone Assay, Binding Assay, Control, Live Cell Imaging

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 2. Loops are unaffected by ZFP143 depletion in mESCs (A and B) Two representative loci averaging clones A/B. From top to bottom are Micro-C maps at 2,000 bp resolution comparing untreated to DZFP143 (3 h), DCTCF (3 h) to DZFP143/DCTCF (3 h), ZFP143 ChIP-seq tracks (cyan), CTCF ChIP-seq tracks (red), and PRO-seq showing plus strand (blue) and minus strand (magenta) for all conditions. Below are zoom-ins on ZFP143-regulated genes showing reduction in PRO-seq signal upon ZFP143 depletion. (A) The Timm13/ Lmnb2 locus. (B) The Rpp30 locus. (C) Aggregate peak analysis (APA)/loop pileup analysis for all loops, cohesin-bound loops, E-P loops, and P-P loops from Hsieh et al.74 and ZFP143-bound loops for untreated, DZFP143 (3 h), DCTCF (3 h), and DZFP143/DCTCF (3 h) conditions in log10 scale with average dot strength (upper left). (D) Scatterplots of loop strengths calculated as in (C) for cohesin loops (red), E-P loops (orange), P-P loops (teal), and ZFP143 loops (cyan) comparing DZFP143 (3 h), DCTCF (3 h), or DZFP143/DCTCF (3 h) to untreated.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: Clone Assay, ChIP-sequencing

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 3. Loops are unaffected at longer ZFP143 depletion timescales in mESCs (A and B) Two representative loci (as in Figures 2A and 2B) in clone D time course. From top to bottom are Micro-C maps at 2,000 bp resolution comparing untreated to DZFP143 (6 h), DZFP143 (12 h), and DZFP143 (24 h); ZFP143 untreated ChIP-seq tracks (cyan); and CTCF untreated ChIP-seq tracks (red). (C) APA/loop pileup analysis for all loops, E-P loops, P-P loops, and ZFP143-bound loops for untreated and DZFP143 (6 h), DZFP143 (12 h), and DZFP143 (24 h) conditions in log10 scale with average dot strength (upper left). (D) Scatterplots of boundary strengths calculated as in (C) for E-P loops (orange), P-P loops (teal), and ZFP143 loops (cyan) comparing DZFP143 (6 h), DZFP143 (12 h), and DZFP143 (24 h) to untreated. (E) Boxplots of the log2 loop strengths plotted in (D) relative to the untreated condition versus ZFP143 depletion time for cohesin (red), E-P (orange), P-P (teal), and ZFP143-bound (cyan) loops. See also Figure S3.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: ChIP-sequencing

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 4. Loops are unaffected by ZFP143 depletion in HEK293T cells (A and B) Two representative loci in HEK293T clone 30. From top to bottom are Micro-C maps at 2,000 bp resolution comparing untreated to DZFP143 (3 h), ZFP143 ChIP-seq tracks (cyan), CTCF ChIP-seq tracks (red), and PRO-seq41 with plus strand (blue) and minus strand (magenta) for untreated and DZFP143 (3 h). Below are zoom-ins on ZFP143-bound genes for the ABL1 locus (A) and the CSRNP2 locus (B). (C) APA/loop pileup analysis for all called loops, E-P loops, P-P loops, and ZFP143-bound loops across untreated and DZFP143 (3 h) conditions in log10 scale with average dot strength (upper left). (D) Scatterplots of boundary strengths calculated as in (C) for E-P loops (orange), P-P loops (teal), and ZFP143 loops (cyan) comparing DZFP143 (3 h) to un- treated. (E) Metaplots over insulation score tracks called at 50 kb resolution comparing untreated (gray) and DZFP143 (3 h) (cyan). (F) Venn diagrams of overlap in genes with ZFP143- or CTCF-bound promoters between mESC and HEK293T. (G) Diagram of mouse and human ZFP143 showing the location of the seven C2H2 zinc-finger domains. Below, identical (teal), similar (cyan), and dissimilar (red) amino acids between species. See also Figure S3.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: ChIP-sequencing, Insulation

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 5. ZFP143 largely binds independently to CTCF (A) Venn diagrams of overlap in CTCF and ZFP143 binding between HEK293T and mESC ChIP-seq. (B) The percentage of ZFP143 and CTCF ChIP-seq peaks overlapping TSS (teal), enhancer (orange), intragenic (red), or intergenic (gray) regions. (C) Scatterplots showing the log2 fold-change of read counts at ChIP-seq peaks between either DCTCF (3 h) and untreated ZFP143 ChIP-seq (left) or DZFP143 (3 h) and untreated CTCF ChIP-seq (right) versus peak size. The number of significantly increased or decreased peaks (red) from DESeq2 (padj % 0.05, fold change R 1.5) is at the top. On the right is a histogram of point density. (D) Metaplots over ChIP-seq peaks for untreated or DCTCF (3 h) ZFP143 ChIP-seq (left) or untreated or DZFP143 (3 h) CTCF ChIP-seq (right). In the left plot, peaks overlapping a CTCF peak (magenta, top) and those not overlapping a CTCF peak (blue, bottom) are shown. (E and F) FRAP curves show normalized intensity over time. Halo-NLS and H2B-Halo controls are gray. Error bars indicate 95% CI. (E) ZFP143 in the untreated (blue) condition versus DCTCF (2–4 h, magenta) averaging clones A/B. (F) CTCF in the untreated (blue) condition versus DZFP143 (2–4 h, magenta). (G and H) Jump displacement CDFs from SPT for an 18 ms time-lag. (G) SPT of ZFP143 comparing DCTCF (2–4 h, magenta) with untreated (blue). (H) SPT of CTCF comparing DZFP143 (2–4 h, magenta) with untreated (blue). (I) State-array SPT (saSPT) showing the posterior distribution versus diffusion coefficient in mm2 s–1 for ZFP143 SPT (left) and CTCF SPT (right). Curves give the mean across traces. The shaded area gives the 95% CI across all movies. Heatmaps show individual traces. Either DCTCF (2–4 h) or DZFP143 (2–4 h) is magenta, and untreated is blue. See also Figures S4 and S5.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: Binding Assay, ChIP-sequencing, Clone Assay, Diffusion-based Assay

Journal: Molecular cell

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

doi: 10.1016/j.molcel.2024.11.031

Figure Lengend Snippet: Figure 6. ZFP143 is a transcriptional regulator of ribosomal and mitochondrial genes (A) Histogram of the distribution of the distance of ZFP143 motifs in ChIP-seq peaks overlapping ±1 kb to a TSS showing a median of 38 bp. (B) Metaplots over PRO-seq signal centered on ZFP143 motifs for the same strand as the ZFP143 motif (blue), and the opposite strand as the ZFP143 motif (magenta). (C) Orientations of motifs in (A) relative to their nearest TSS. (D and E) Volcano plots showing –log10 of the padj value versus the log2 fold-change of read counts of PRO-seq signal at genes. Genes with a fold-change greater than two are green, genes with a padj value less than 0.001 are blue, and genes significant in both are red. The number of significantly changed genes evaluated by DESeq2 is above. (D) DZFP143 (3 h). (E) DCTCF (3 h). (F) Metaplots over gene bodies showing average PRO-seq signal in the untreated (left) versus DZFP143 (3 h, right) conditions. Genes with ZFP143-bound promoters (magenta, upper). Non-ZFP143-bound genes (blue, bottom). (G) The percentage of significantly changing expression genes bound by either ZFP143 (cyan), CTCF (red), both (teal), or neither (gray) in PRO-seq evaluated by DESeq2. (H) Venn diagrams of overlaps in genes significantly changing in the DZFP143 (3 h, cyan), DCTCF (3 h, red), or DZFP143/DCTCF (3 h, teal) conditions. (I) Significant gene sets versus normalized enrichment score for GO-term enrichment analysis of significantly changed genes in the DZFP143 (3 h) condition. The size of the circle corresponds to the set size, and points are colored by log10 padj value. See also Figure S6.

Article Snippet: Due to the findings by Magnitov and Maresca et al. in their 6 Molecular Cell 85, 1–15, January 2, 2025 companion paper that a commonly used ZFP143 antibody from Proteintech cross-reacts with CTCF, we used an antibody from Novus Biologicals, which does not show cross-reactivity.95 Using this antibody, we found that for all clones, >90% of ZFP143 peaks responded to the depletion, indicating no cross-reactivity (Figure S4E).

Techniques: ChIP-sequencing, Expressing

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 1. Overview of the design and dual-functionality of the TRE-Lox system. (a) Overall structure of the 5′ end of the murine cathepsin D (CatD) gene (CTSD) and its promoter region (PCTSD, dark gray), indicating the relative position of the two gRNAs (black arrows) used for CRISPR/Cas9- assisted homologous recombination. Note the placement of the TATA box (TATA) very close to the main transcription start site (TSS) (right-angle arrow), the presence of the initiation codon (ATG, dashed white line) within Exon 1 (Ex 1, light gray), and the presence of a splice donor (SD) and splice acceptor (SA) flanking Intron 1 (black line). (b) Structure of the TRE-Lox knock-in (KI) insert, illustrating the relative positions of the two tet-operons (tetO2, green) and one LoxP site (LoxP, light blue) within the 5′ untranslated region (5′UTR) and, within Intron 1, a tetracycline response element (TRE) comprised of seven tetO repeats (tetO7) and the second LoxP site. The relative placement of the puromycin resistance cassette (Puror, purple) flanked by two FRT sites (FRT, dark blue), which is excisable by Flp recombinase, is depicted using a curly bracket. (c) Downregulation of CTSD via the action of rtTRKRAB acting on the TRE-Lox insert. In the presence of Dox (red triangles), rtTRKRAB binds to the tetO repeats within both the 5′UTR and Intron 1, triggering methylation of histones in a radius of 2–3 kb, thereby remodeling the chromatin and silencing the CTSD gene. (d) Genetic deletion of CTSD via the action of Cre recombinase on the TRE-Lox insert. The figure depicts the end result of Cre-mediated recombination of the TRE-Lox KI insert, which causes removal of the initiation codon, the first portion of the coding region of Exon 1 encoding the signal peptide of CatD, and the 5′ end of Intron 1.

Article Snippet: Relevant regions (and their sources) were as follows: the 3′ end of Exon 1 and the 5′ portion of Intron 1 of murine CTSD (from C57Bl6/J mouse tail DNA); tetO2 (from Addgene plasmid #113892 [41]); TRE 3G and, separately, an FRT-flanked puromycin resistance cassette (both from Addgene plasmid #156430 [42]).

Techniques: CRISPR, Homologous Recombination, Knock-In, Methylation

Journal: PLoS Genetics

Article Title: Two cis -regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

doi: 10.1371/journal.pgen.1009119

Figure Lengend Snippet: (A) Supershift EMSA was performed using CTCF antibody. CTCF antibody (1 μg) was incubated with 0.5 μg recombinant human CTCF protein prior to the addition of the binding buffer and probe. G and A represent labelled probes containing the white eggshell and blue eggshell allele, respectively, and G cold and A cold represent cold probes containing the white eggshell and blue eggshell allele, respectively. (B) ChIP-qPCR analysis of CTCF binding on variation M5 in uteruses from blue-eggshelled and white-eggshelled ducks. Chromatin extracts from four individuals of each genotype were immunoprecipitated with CTCF antibody. ChIP-enriched DNA was quantified by qPCR with primers specific for variation M5. Rabbit IgG was used as a negative control. Values of immunoprecipitated samples were normalized to that of the input DNA. Data are presented as mean±SD from four independent individuals.

Article Snippet: CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein (Novus biologicals, Littleton, USA) prior to addition of the binding buffer and probe.

Techniques: Incubation, Recombinant, Binding Assay, ChIP-qPCR, Immunoprecipitation, Negative Control

Journal: PLoS Genetics

Article Title: Two cis -regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

doi: 10.1371/journal.pgen.1009119

Figure Lengend Snippet: (A) Effect of knockdown of CTCF on the promoter activity of causative sites. The pGL3-Basic vectors containing the blue eggshell or white eggshell alleles inserted in their upstream region were transfected into DEF cells 6 hours after transfection of CTCF siRNA1 or negative control siRNA (scramble siRNA) or without siRNA. Cells were collected for luciferase activity analysis 30 hours after siRNA transfection. The group only transfected with the blue eggshell or white eggshell vector was used as control. Data represent the mean±SD from three biological repeats per vector. ** indicates P<0.01. (B) Schematic map of CpGs sites used in the DNA methylation analysis. The causative site M5 was referred as position +1. The black bars represented the CpG dinucleotides upstream or downstream of the predicted CTCF binding sites which were marked in grey background. (C) Heatmap of uterine DNA methylation levels of CpG sites in region A and B of blue-eggshelled and white-eggshelled ducks. The DNA methylation level was determined by sequencing 8 clones of bisulfite treated genomic DNA for each individual. The DNA methylation level of each CpG site was represented by the average methylation of 8 clones. The DNA methylation level from 0 to 1 is shown in the color from blue to red.

Article Snippet: CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein (Novus biologicals, Littleton, USA) prior to addition of the binding buffer and probe.

Techniques: Knockdown, Activity Assay, Transfection, Negative Control, Luciferase, Plasmid Preparation, Control, DNA Methylation Assay, Binding Assay, Sequencing, Clone Assay, Methylation

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased TREK1 expression upon Aβo treatment, which is markedly reduced by co-treatment with the calcium chelator BAPTA-AM. b, Quantification of TREK1 fluorescence intensity following Aβ42o and/or BAPTA-AM treatment (n=61-90 Cells; **p < 0.01, ####p < 0.0001; one-way ANOVA with Šidák’s test). c, Representative images showing that TREK1 increase induced by Aβ42o is attenuated by the AC1 inhibitor ST034307. d, Quantification of TREK1 fluorescence intensity following Aβ42o and/or ST034307 treatment (n=23-31 cells; **p < 0.01, ###p < 0.001; one-way ANOVA with Šidák’s test). e, Representative images showing TREK1 expression is decreased in Aβ42o treated neurons transfected with AC1-specific siRNA but not with scrambled (Sc) siRNA. f, Quantification of TREK1 fluorescence intensity following Aβ42o treatment with AC1 knockdown (n=71-128 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). g, Representative images showing TREK1 expression is reduced in Aβ42o-treated neurons transfected with AC8-specific siRNA, but not with scrambled siRNA. h, Quantification of TREK1 fluorescence intensity following Aβ42o treatment with AC8 knockdown (n=14-19 cells; ****p < 0.0001, unpaired t-test). i, Treatment with the cAMP analog 8-CPT-cAMP increases TREK1 expression in primary neurons. j, Quantification of TREK1 fluorescence intensity following 8-CPT- cAMP treatment (n=57-62 cells; ***p < 0.001, unpaired t-test). k, Treatment with forskolin, a cAMP activator, mimics Aβ42o by increasing TREK1 expression in primary neurons. l, Quantification of TREK1 fluorescence intensity following forskolin treatment (n=146-242 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). m, Schematic representation illustrating that Aβ42o-induced TREK1 upregulation is mediated by calcium influx via the AC1/AC8–cAMP signaling pathway. Data are presented as mean ± SEM. 3-5 independent cultures per group were used.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Expressing, Fluorescence, Transfection, Knockdown

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased TREK1 expression in Aβ42o-treated neurons, which is decreased upon co-treatment with the PKA inhibitor KT5720. b, Quantification of TREK1 fluorescence intensity following Aβ42o and/or KT5720 treatment (n = 52-70 cells; ****p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). c, Representative images showing that TREK1 increase induced by Aβ42o is attenuated by another PKA inhibitor H89. d, Quantification of TREK1 fluorescence intensity following Aβ42o and/or H89 treatment (n = 43–49 cells; **** p < 0.0001, ####p < 0.0001; one-way ANOVA with Šidák’s test). e, Schematic representation of the TREK1 locus on chromosome 1q41 showing predicted promoters (blue boxes; P1–P3) and ENCODE-annotated CTCF binding sites (red boxes; accession IDs indicated). Genomic positions are indicated relative to the transcription start site (TSS). ReMap ChIP-seq and density tracks demonstrate experimental support for the predicted binding sites, with peaks at site E1421793 located proximal to the promoter (P1). Motif analysis using JASPAR confirmed the presence of a consensus CTCF motif within this region (right panel), with associated FIMO statistics (score, p-value, and q-value). The identified site (highlighted in red) spans chr1:215076354–215076685 (band 1q41), has a genomic size of 332 bp, and is classified as “CTCF-bound” in ENCODE. f, Representative images showing decreased TREK1 expression in Aβ42o treated neurons transfected with CTCF-specific siRNA compared to scrambled (Sc) siRNA. g, Quantification of TREK1 fluorescence intensity following Aβ42o and/or CTCF knockdown (n = 37–76 cells; **** p < 0.0001, #### p < 0.0001; one-way ANOVA with Šidák’s test). h, Representative images showing decreased TREK1 expression in hippocampal neurons of 3xTg mice following intrahippocampal injection of CTCF shRNA lentivirus compared to control. i, Quantification of TREK1 fluorescence intensity in 3xTg mice after CTCF knockdown (n = 13–19 sections; ****p < 0.0001; unpaired t-test). j, Schematic representation of the PKA–CTCF signaling axis in regulating Aβ42- induced TREK1 expression. Data are presented as mean ± SEM. 3-5 independent cultures or animals per group were used.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Expressing, Fluorescence, Binding Assay, ChIP-sequencing, Transfection, Knockdown, Injection, shRNA, Control

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative calcium imaging traces from control and Aβ42o treated neurons showing that Aβ42o increases spontaneous calcium transient frequency. This hyperexcitability is further enhanced by the TREK1 inhibitor spadin and suppressed by the TREK1 activator BL-1249. b, Quantification of calcium event frequency upon treatment with Aβ42o/spadin/BL-1249 (n = 512–1002 cells; ****p < 0.0001, ##p < 0.01, †p < 0.05; one-way ANOVA with Šidák’s test). c, Representative FluoVolt traces measuring membrane potential fluctuations manifest enhanced neuronal activity in the presence of TREK1 inhibitor spadin and suppressed neuronal activity by the TREK1 activator BL-1249 compared to Aβ42o treatment alone. d, Quantification of potential spike frequency upon treatment with Aβ42o/spadin/BL- 1249 (n = 31–105 cells; **p < 0.01, #p < 0.05, ††p < 0.01; one-way ANOVA with Šidák’s test). e, Representative calcium traces from neurons treated with Aβ42o along with scrambled (Sc) siRNA, KCNK2 siRNA, or a KCNK2 overexpression (OE) construct. f, Quantification of calcium event frequency upon knocking down KCNK2 in the presence of Aβ42o treatment (n = 49–67 cells; *p < 0.05; unpaired t-test). g, Quantification of calcium event frequency upon overexpressing KCNK2 in the presence of Aβ42o treatment (n = 37–48 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). h, Representative patch-clamp recordings of action potentials in control, Aβ42o, and Aβ42o + spadin treated neurons showing exacerbated action potential firing with TREK1 blockade. i, Quantification of action potential frequency following Aβ42o and/or spadin treatment (n = 17 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). j, Resting membrane potential (RMP) is more depolarized in Aβ42o treated neurons compared to control, and further depolarizes in presence of spadin with Aβ42o (n = 19 cells; ****p < 0.0001, #p < 0.05; one-way ANOVA with Šidák’s test). k, Representative traces showing excitatory postsynaptic current (EPSC) frequency is increased in neurons treated with Aβ42o + spadin compared to Aβ42o alone. l, Quantification of EPSC frequency (n = 19 cells; *p < 0.05, ##p < 0.01; one-way ANOVA with Šidák’s test). m, Quantification of EPSC amplitude (n = 19 cells). n, Representative traces showing Inhibitory postsynaptic current (IPSC) frequency is decreased in neurons treated with Aβ42o+spadin compared to Aβ42o alone. o, Quantification of IPSC frequency (n = 10 cells; **p < 0.01, #p < 0.05; one-way ANOVA with Šidák’s test). p, Quantification of IPSC amplitude (n = 10 cells). q, Representative ex vivo calcium imaging heat map from hippocampal slices of 3xTg mice injected with TREK1 shRNA lentivirus showing elevated calcium activity compared to sc shRNA-injected mice. r, Representative calcium imaging traces demonstrating increased calcium transient frequency following TREK1 knockdown. s, Quantification of calcium event frequency in TREK1 knockdown mice compared to sc shRNA-injected mice (n = 13–27 cells; *p < 0.05; unpaired t-test). Data are presented as mean ± SEM from 3-5 independent cultures.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Imaging, Control, Membrane, Activity Assay, Over Expression, Construct, Patch Clamp, Ex Vivo, Injection, shRNA, Knockdown

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased VGLUT1 intensity in the hippocampus of 3xTg mice 15 days after intrahippocampal injection with TREK1 shRNA lentivirus compared with vehicle-injected controls. b, Representative images showing decreased VGAT intensity under the same TREK1 knockdown conditions. c, Quantification of VGLUT1 fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 67–76 sections; ****p < 0.0001; unpaired t-test). d, Quantification of VGAT fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 60–63 sections; ****p < 0.0001; unpaired t-test). e, Quantification of Excitatory/inhibitory (E/I) ratio, calculated as VGLUT1/VGAT intensity, is markedly elevated in TREK1 knockdown mice compared with vehicle injected controls (n = 3– 4; **p < 0.01; unpaired t-test). f, Representative immunofluorescence images demonstrating enhanced Aβ deposition in the hippocampus of TREK1 knockdown 3xTg mice compared with vehicle-injected controls. g, Quantification of Aβ fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 50 sections; *p < 0.05; unpaired t-test). h, Quantification showing a significant decrease in MAP2 intensity in the hippocampus of 3xTg mice injected with TREK1 shRNA compared with scrambled (Sc) shRNA controls (n = 159–166 sections; ****p < 0.0001; unpaired t-test). i, Schematic representation illustrating the effects of TREK1 knockdown on excitatory/inhibitory balance. Data are expressed as mean ± SEM from 3–4 mice per group.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Immunofluorescence, Injection, shRNA, Knockdown, Fluorescence

Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: The diagram summarizes the signaling cascade by which Aβ42 induced neuronal hyperexcitability drives TREK1 upregulation via calcium influx and the AC1/AC8–cAMP–PKA–CTCF axis. TREK1 upregulation decreases neuronal excitability, limits excitatory/inhibitory balance, thereby improves neuronal health in 3xTg mice.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: