Journal: Genome biology

Article Title: BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

doi: 10.1186/s13059-024-03175-0

Figure Lengend Snippet: Fig. 7 Opening of chromatin by BORIS facilitates binding of other transcriptional factors. a Scatter plot displaying the enrichment of 190 TF motifs at CTCF/BORIS binding sites that reprogrammed into active promoters, compared to transcriptionally silent CTCF/BORIS sites in NIH3T3 + BORIS (clone#2) cells. b MAZ and MXI1 motifs are significantly enriched at CTCF/BORIS binding sites converted into active promoters. c Genome browser view illustrates the recruitment of TBP, HCFC1, MXI1, and MAZ proteins at the CTCF site within the Rbpjl promoter, activated by BORIS binding in NIH3T3 + BORIS (clone#2) cells. The activated promoter is highlighted by a red open box. d Left panel: Scatter plot of normalized read counts (log10) for HCFC1 occupancy at the combined set of HCFC1 binding sites (46,287) in NIH3T3 + BORIS (clone#2) cells compared to the same genomic sites in EV cells. Right panel: Heatmap of CTCF (red), BORIS (blue), and HCFC1 (brown) occupancy at the 19,519 HCFC1 sites from the left panel (connected by red arrow). e TF motifs enriched at HCFC1 peaks (60 bp around the summit of peak) in NIH3T3 + EV versus NIH3T3 + BORIS (clone#2) cells. f Heatmap of BORIS (blue), TBP (purple), HCFC1 (brown), MXI1 (orange), and MAZ (green) occupancy at the 5871 CTCF/BORIS binding sites converted into active promoters in NIH3T3 + BORIS (clone#2) cells compared to NIH3T3 + EV cells from Fig. 4h. g Summary of epigenetic reprogramming: BORIS binding recruits SRCAP, which replaces H2A histone with H2A.Z, leading to the opening of chromatin around CTCF sites. This, in turn, attracts other TFs to bind and stimulate transcription, resulting in the conversion of transcriptionally inert CTCF sites into active promoters

Article Snippet: Rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), rabbit polyclonal against HCFC1 (Novus Biologicals, NB100-68209), goat affinity-purified polyclonal antibody against Mxi1 (R&D Systems, AF4185), rabbit monoclonal to TATA-binding protein TBP (Abcam, ab220788), rabbit polyclonal anti-PHF8 antibody (Bethyl Laboratories, A301-772A), rabbit polyclonal to MAZ (Abcam, ab85725), rabbit polyclonal antibody against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3) (Diagenode, C15410195), rabbit polyclonal antibody against histone H3, trimethylated at lysine 36 (H3K36me3) (Diagenode, C15410058), rabbit polyclonal antibody against histone H2A.Z (Abcam, ab4174), rabbit polyclonal to Histone H3 (acetyl K27) (Abcam, ab4729), rabbit polyclonal to Histone H3 (tri methyl K4) (Abcam, ab8580), anti-RNA Polymerase II Antibody, CTD Antibody, clone 8WG16 (Millipore, 05–952-I-100UG).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: Large mega-domain deletions on the X chromosome identify HCFC1 as a potential XCI activator. a , Targeting and differentiation strategies. Rnf12 +/- cells were transiently transfected with CRISRPR-Cas9 and gRNAs targeting different locations on the X chromosomes. Correctly targeted clones were differentiated for several days. b , X chromosome scheme depicting evolutionary strata 1, 2, 3, 4 and the pseudo-autosomal region (PAR) , . The locations of Xist and Rnf12 are depicted with a black and grey line, respectively, while the deleted Rnf12 allele on the 129 chromosome is also shown. The different deletions (ΔA to ΔK) are shown with different colors underneath. c , Relative Xist expression at day 8 of differentiation of WT, Rnf12 +/- and the different Rnf12 +/- cell lines with X chromosomal deletions, normalized to Rnf12 +/- . Average ± SD, n=2 to 5 biological replicates. A one-sided Welch’s t-test was used to test significant differences between Rnf12 +/- and WT or deletion cell lines. P-values were corrected for multiple testing with the Benjamini-Hochberg method. d , Zoom-in on a specific region of ΔE. The locations of different overlapping deletions (ΔE1, ΔE2, ΔE3) are shown with black lines; the smaller incremental deletions (ΔE2A, ΔE2B, ΔE3A, ΔE3B) are shown in green. The blue rectangle highlights the location of Hcfc1 and Irak1 . e , Expression heatmap of X-linked genes, sorted by genomic location and split by allele (129 or Cast) for 2 WT, 2 Rnf12 +/- , 1 ΔE2A, 2 ΔE2B, 2 ΔE3A, 2 ΔE3B and 2 ΔE clones. Notice the deleted genes per clone on the 129 alleles. f , Relative Xist expression at day 0 and day 8 of WT, Rnf12 +/- , ΔE and smaller ΔE deletion cell lines. Average expression ± SD; n=2 (WT, Rnf12 +/- and ΔE2A lines), n=4 (ΔE2B, ΔE3A and ΔE3B lines) and n=5 (ΔE lines) biological replicates. A one-sided Welch’s t-test was used to test significant differences per day of differentiation between Rnf12 +/- and WT or deletions cell lines. P-values were corrected for multiple testing with the Benjamini-Hochberg method. g , Volcano plot portraying gene expression changes between ΔE2B and Rnf12 +/- cells. Downregulated and upregulated genes in ΔE2B are shown in blue and red, respectively. Notice Xist is depicted on the left, as the most significant downregulated gene. h , Same as g but for ΔE3B vs Rnf12 +/- cells. i , Expression (TPM) of Hcfc1 (green), Irak1 (grey), Rnf12 (blue) and Xist (red) in ΔXic Cast ESCs from Pacini et al., 2021 . P-values were calculated between day 0 and 1 for Hcfc1 (green) and Irak1 (grey) using one-sided independent t-tests and corrected for multiple testing with the Benjamini-Hochberg method.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Transfection, Clone Assay, Expressing, Gene Expression

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: Megadomain deletion confirmation by PCR and WGS. a , Overview of the locations of the different deletions generated in this study. Different X-linked length polymorphisms between 129 and Cast are shown (DX amplicons). b-c, PCR analysis of different DX amplicons for the different deletions. d , Location of the different DX amplicons with respect to the deleted regions, and length polymorphism analysis inside and around several incremental deletions within region E. e , Scatter plots of allelic ratios along the X-chromosome from shallow whole-genome sequencing for different ΔE, ΔE2A, ΔE2B, ΔE3A and ΔE3B clones (left). Y-axis represents the log 2 (fold change) between the 129 and Cast alleles for each window. Windows with significant signal differences between alleles are highlighted in green. The zoomed-in plots on the right show the region around 67-77 Mb, with the Hcfc1 -containing window indicated by a star.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Generated, Sequencing, Clone Assay

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: Deletions containing Hcfc1 show reduced Xist upregulation but can differentiate. a , DNA FISH analysis on metaphase spreads of the parental and three ΔE clones detecting Hprt and Xist in green, and Mecp2 in red. Notice red is inside the deleted region. White arrowheads indicate chromosome X. Arrowheads with an asterisk in ΔE clone 3 indicate the signal within an interphase nucleus. b , Representative images of Xist RNA FISH (red) in WT, Rnf12 +/- and three ΔE clones at day 8 of differentiation. c , Quantification of b , average number of cells with a Xist cloud ± SD, n=3 biological replicas, n=201-231, statistical significance was calculated using a two-sided Welch T-test and corrected for multiple testing using the Benjamini-Hochberg procedure. P-values are indicated, ns = not significant. d , Allelic gDNA PCR of Tsix in WT, Rnf12 +/- and 3 independent ΔE clones on day 8 of differentiation to confirm the presence of two X chromosomes. Average ± SD, n=1-3 biological replicates. e , Relative Xist expression in WT, Rnf12 +/- , ΔE, ΔE1, ΔE2 and ΔE3 ESCs at day 0 and 8 of differentiation. A one-sided Welch’s t-test was used to test significant differences between Rnf12 +/- and WT or deletion cell lines. P-values were corrected for multiple testing with the Benjamini-Hochberg method. f , Heatmap of gene expression (in TPMs) for pluripotency markers ( Pou5f1 , Nanog , Zfp42 / Rex1 , Klf4 ) and differentiation markers ( Kdr / Flk-1 , Gata4 , Gata6 , T and Nodal ) across WT, Rnf12 +/- and ΔE, ΔE2A, ΔE2B, ΔE3A and ΔE3B clones. g , Violin plot of allelic ratios of X-linked gene expression in Rnf12 +/- , ΔE, ΔE2B and ΔE3B. The box plots display the median (black line), the interquartile range (box limits) and 1.5x of the interquartile range (whiskers). Dashed line indicates ratio of 0.5 as expected ratio without silencing. P-values were calculated using a two-sided Mann-Whitney U test and corrected for multiple testing using the Benjamini-Hochberg approach. h , Expression (TPM) of Hcfc1 (green), Irak1 (grey), Rnf12 (blue) and Xist (red) in ΔXic B6 ESCs from Pacini et al., 2021 . P-values were calculated between day 0 and 1 for Hcfc1 (green) and Irak1 (grey) using one-sided independent t-tests and corrected for multiple testing with the Benjamini-Hochberg method.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Clone Assay, Expressing, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: HCFC1 is an XCI activator. a , HCFC1 rescue and differentiation strategy of control (Ctrl; parental Rnf12 +/- ), ΔE2B and ΔE3B ESCs. b , RT-qPCR analysis of Hcfc1 expression upon Hcfc1 rescue in pools of transfected cells after 4 days of differentiation of in Ctrl (parental Rnf12 +/- ), ΔE2B and ΔE3B. The average of n=2 biological replicates is shown. c , RT-qPCR analysis of Xist expression in pools of transfected cells after 4 days of differentiation. The average of n=2 biological replicates is shown. d , 2xFLAG-V5-FKBP12 F36V tagging strategy of HCFC1 and differentiation schemes with different dTAG-13 treatment windows. e , Western blot of the C-terminal fragments of HCFC1 upon dTAG-13 treatment in undifferentiated ESCs. f , RT-qPCR analysis of Xist expression at different timepoints after start of dTAG-13 treatment and differentiation normalised to DMSO control; see . A two-sided Welch’s t-test was used to assess significant differences between DMSO- and dTAG-13 treated cells. P-values were corrected for multiple testing using the Benjamini-Hochberg method. g , Xist RNA-FISH analysis in HCFC1-FKBP ESCs differentiated for 3 days and treated with dTAG-13 for the last 48h vs DMSO and parental ESCs. h , Quantification of g . Average ± SD, n=3-6 biological replicates, n=157-236. A two-sided Welch’s t-test was used to assess significant differences between DMSO- and dTAG-13 treated cells. P-values were corrected for multiple testing using the Benjamini-Hochberg method. i , Gene expression levels of several genes located in the XIC determined by RNA-seq analysis of HCFC1-FKBP ESCs differentiated for 3 days and treated with dTAG-13 for the last 48h. Shown in green are genes residing in the Xist TAD and involved in its activation. In red, genes located in the Tsix TAD and involved in Xist repression. Average TPMs normalized to 48h DMSO control ± SD, n= 3 biological replicates. P-values were calculated using DESeq2, which corrects for multiple testing using the Benjamini-Hochberg method. j , Violin plot of allelic ratios of X-linked gene expression in the DMSO and dTAG-13 conditions. The box plots display the median (black line), the interquartile range (box limits) and 1.5x of the interquartile range (whiskers). Dashed line indicates ratio of 0.5 as expected ratio without silencing. P-value was calculated using a two-sided Mann-Whitney U test.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Control, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Gene Expression, RNA Sequencing, Activation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: HCFC1 rescue and conditional knockout confirmation. a , Relative Hcfc1 expression in control ( Rnf12 +/- ), ΔE2B and ΔE3B ESCs upon stable integration, Hcfc1 expression vs control plasmid in different individual undifferentiated clones; Average ± SD, n=4-5 independent clones. b , Relative Xist expression in Control ( Rnf12 +/- ), ΔE2B and ΔE3B upon stable integration, Hcfc1 expression vs control plasmid at day 4 of differentiation in different individual clones; Average ± SD, n=4-5 independent clones. c , Same as in b , but for differentiation marker Nestin . d , PCR analysis of HCFC1-FKBP ESCs, top. DX95 PCR to control for the presence of both X chromosomes, bottom. e , HCFC1 C-term and V5 WB in HCFC1-FKBP ESCs after treatment with dTAG-13 or DMSO control in the ESC state. Actin is used as loading control. f , Expression of several pluripotency markers at day 3 of differentiation after 48h of dTAG-13 or DMSO treatment. TPM values are normalized to the average expression in the DMSO condition for each gene. P-values were calculated using DESeq2, which corrects for multiple testing using the Benjamini-Hochberg method g , Same as in f , but for genes involved in Xist regulation. h , Schematic of HCFC1-FKBP ESCs with a doxycycline-inducible Xist promoter and strategy to induce Xist expression in the ESC stage in the presence of dTAG-13. i , RT-qPCR analysis of Xist expression in HCFC1-FKBP ESCs upon 24h of dox treatment and/or 30h dTAG-13 treatment. Average ± SD, n=3 biological replicates. j , Allelic expression of X-linked gene Rnf12 in HCFC1-FKBP ESCs upon 24h Dox and/or 30h dTAG-13 treatment. Average ± SD, n=3 biological replicates. k , Allelic expression of XCI escapee Kdm6a upon forced Xist expression (+Dox) for 24h in the presence of dTAG-13 or DMSO. For (i–k), p-values from two-sided Welch’s t-test between DMSO and dTAG-13 treatment, corrected using the Benjamini–Hochberg procedure.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Knock-Out, Expressing, Control, Plasmid Preparation, Clone Assay, Marker, Quantitative RT-PCR

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: HCFC1 binding correlates with promoters, H3K4me3 deposition and YY1 binding. a , Heatmap of HCFC1, YY1, H3K4me3, DPY30, PolIISer5 and INTS11 enrichment at HCFC1 peaks in undifferentiated ESCs (left). The right panels show HCFC1 and H3K4me3 enrichment at day 3 of differentiation at HCFC1 peaks identified at day 0. b , HCFC1-only and YY1-only and shared HCFC1-YY1 peak distribution at different genomic locations at day 0. c , Venn diagram with the overlap of HCFC1 and YY1 peaks in promoter regions at day 0 and 3 of differentiation. d , Correlation between HCFC1 binding at day 0 and day 3 in promoter regions, with r representing the Pearson correlation coefficient. e , Genome browser views showing examples of HCFC1-YY1 overlapping peaks in promoter regions. f , MA plot depicting H3K4me3 enrichment changes upon dTAG-13-mediated removal of HCFC1. In red, peaks with increased H3K4me3 enrichment; in grey, unaffected peaks and in blue, peaks with reduced H3K4me3 enrichment. g , Violin plots showing H3K4me3 changes in promoter regions of differentially expressed genes from the RNA-seq comparison between HCFC1-FKBP dTAG-13 and DMSO. P-values were calculated using two-sided Mann-Whitney U tests, corrected for multiple testing using the Benjamini-Hochberg approach. h , Heatmaps showing differences in H3K4me3 and INTS11 enrichment at H3K4me3 peaks that are lost upon dTAG-13-mediated removal of HCFC1 and unaffected H3K4me3 peaks. For both H3K4me3 and INTS11, the fold change between dTAG-13 and DMSO treatment is shown. HCFC1 binding (day 3) and YY1 binding (day 0) are displayed on the right. For the unaffected peaks, a random subset of 4,991 peaks is shown.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Binding Assay, RNA Sequencing, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: HCFC1 binding sites analysis and genome-wide H3K4me3 and INTS11 recruitment upon dTAG-13-mediated depletion of HCFC1. a , Genomic annotation of HCFC1 peaks at day 0 and day 3 of ESC differentiation. b , PCR analysis confirming proper integration of HCFC1-V5- and the YY-FKBP-tags and presence of both X chromosomes (DX95). c , Feature enrichment analysis at HCFC1 peaks at day 3 of differentiation. Features are grouped by over- and underrepresentation, and the top 10 with the lowest p-values are labeled. d , Motif enrichment analysis at HCFC1 peaks at day 3 of differentiation. The 10 most significantly enriched motifs are labeled. e , Western blot analysis of H3K4me3 in HCFC1-FKBP cells treated with DMSO or dTAG-13 (48h) at day 3 of differentiation. f , Quantification of global H3K4me3 WB signal in Ctrl and HCFC1-FKBP cells treated with DMSO or dTAG-13 (48h) at day 3 of differentiation. ns, not significant, p-values were calculated using a two-sided Welch’s t-test and corrected via Benjamini-Hochberg. g , Heatmap of H3K4me3 and INTS11 enrichment at H3K4me3 peaks lost upon HCFC1 depletion, and at unaffected peaks (random subset of 4,991 peaks). h , MA plot showing gene expression changes upon dTAG-13-mediated depletion of HCFC1. Genes are colored based on significant upregulation (red), downregulation (blue) or no change (grey). j , Heatmap of H3K4me3 and INTS11 changes upon HCFC1 depletion, as well as HCFC1 and YY1 binding and H3K4me3 and INTS11 enrichment in the DMSO condition, at promoters of upregulated, unaffected and downregulated genes.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Binding Assay, Genome Wide, Labeling, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: YY1 interacts with the Integrator complex. a , YY1 interactome identified by FLAG pull-down and mass spectrometry. Volcano plot showing proteins enriched in YY1 pull-downs compared to control. Known members of the Integrator (light blue), INO80 (dark blue), and COMPASS (green) complexes are highlighted. Selected proteins, including YY1 itself and HCFC1, are labeled in black. Dashed lines denote thresholds for significance (p < 0.05, log₂ fold change > 1). Data are based on two biological replicates for FLAG-YY1 and four biological replicates for the control. b , Percentages of shared YY1-INTS11, YY1-only or INTS11-only peaks at different genomic locations at day 0. c , Venn diagram showing the overlap of YY1 and INTS11 binding in promoter regions. d , Generation strategy of the FKBP12 F36V -HA-Yy1:2xFLAG-V5-Hcfc1 compound knockin ESC lines by homologous recombination. e , Stacked barplot showing percentages of H3K4me3, HCFC1 and INTS11 peaks that significantly lost or gained enrichment or remained unchanged, upon HCFC1 or YY1 loss. f, MA plot depicting H3K4me3 enrichment changes upon dTAG-13-mediated removal of YY1. In red, peaks with increased H3K4me3 enrichment; in grey, unaffected peaks and in blue, peaks with reduced H3K4me3 enrichment. g , Scatter plot comparing motif enrichment between gained and lost HCFC1 peaks shown in e . Each point represents a motif, plotted by its -log₁₀(p-value) in both analyses, and motifs with the lowest p-values in either condition are labeled. h , Barplot showing the number of up- and downregulated genes with reduced HCFC1 binding in the promoter region upon YY1 depletion. P-value was calculated using a Pearson’s chi-squared (χ²) goodness-of-fit test, based on the overall proportion of up- and downregulated genes. i , Same as h , but for promoter regions with reduced INTS11 binding. j , Heatmap showing changes in INTS11 and H3K4me3 enrichment upon dTAG-13-mediated depletion of YY1 or HCFC1, alongside YY1 and HCFC1 binding at INTS11 peaks. Peaks are grouped based on whether INTS11 enrichment is reduced (top) or unaffected (bottom) by YY1 depletion.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

Techniques: Mass Spectrometry, Control, Labeling, Binding Assay, Knock-In, Homologous Recombination

Journal: bioRxiv

Article Title: HCFC1 and YY1 mediate recruitment of COMPASS and Integrator to initiate X chromosome inactivation

doi: 10.1101/2025.09.18.677040

Figure Lengend Snippet: HCFC1 binds to and regulates the expression of several non-coding genes in the XIC. a , Genome browser views showing ChIP-seq tracks of H3K4me3 in HCFC1-depleted or YY1-depleted versus DMSO controls at day 3 of differentiation, HCFC1 ChIP-seq in YY1-depleted versus its DMSO control at day 3 of differentiation, INTS11 ChIP-seq in YY1-depleted versus its DMSO control at day 3, and YY1 and HCFC1 ChIP-seq in female ESCs at day 0 around the Tsix , Xist , Jpx and Ftx promoter regions. b, Relative expression of Tsix, Xist , Jpx , Ftx, Hcfc1 and pluripotency factor Rex1 at day 3 of differentiation of YY1-FKBP ESC cells normalised to DMSO control. A two-sided Welch’s t-test was used to test significant differences between DMSO- and dTAG-13 treated cells. P-values were corrected for multiple testing using the Benjamini-Hochberg method. c, Gene-regulatory network controlling Xist expression. RNF12 promotes YY1 binding by targeting the Xist repressor REX1 for proteasomal degradation. HCFC1/COMPASS and YY1 are both required for H3K4me3 catalysis at the Xist regulatory element. YY1 also recruits Integrator to activate transcription of Xist cis-regulatory genes within the XIC. d , Dose-dependent activation of Xist expression. Elevated levels of X-linked activators in XX cells raise the probability for Xist transcription initiation above threshold; this probability is reduced in Rnf12 +/− or Hcfc 1 +/− cells and further diminished in double-heterozygous Rnf12 +/− ; Hcfc 1 +/− cells.

Article Snippet: The membrane was washed 3× with 1×TBST (0.02M Tris-HCl, 0.15M NaCl, 0.1% Tween-20, pH 7.6), incubated with the appropriate primary antibodies, HCFC1 Carboxy-terminal Antigen antibody (Cell signaling, 50708S, 1:1000), V5 antibody (Invitrogen, R96025, 1:5000), YY1 antibody (Santa Cruz, sc-7341, 1:200) and/or H3K4me3 antibody (Sigma-Aldrich, 05-1339, 1:1000).

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