Journal: bioRxiv

Article Title: Epigenetic de-repression of basal cell metaplasia in aging AT2 cells is a risk factor for idiopathic pulmonary fibrosis (IPF)

doi: 10.64898/2026.06.09.731212

Figure Lengend Snippet: (A.) Single-cell transcriptomic-derived dimension reduction plot (UMAP) of AT2 cells from 2 young (22F, 28M) and 2 old donors (67M, 79F) highlighting higher NF-kB activity scores in old donors, as quantified with decoupleR. (B.) Heatmap of transcription factor activity scores between most differentially enriched drivers between NF-kB-high and NF-kB-low AT2 cell clusters across all ages. (C.) Volcano plot of differentially expressed genes between old and young AT2 cells across all donors. Highlighted are several inflammatory regulators and cytokines that are more highly expressed in old donors. (D.) Heatmap of z-scores from pairwise upstream regulator Ingenuity Pathway Analysis (IPA) of differentially expressed genes between and young AT2 cells. (E.) Experimental setup for in vitro IL1β stimulation assays. AT2 cells isolated from young donors were treated with IL1β for 3 days and trans-differentiation was then induced either by mesenchymal co-culture or by inducing HIF with roxadustat in mesenchyme-free conditions. (F.) Violin plots of scRNA-seq data showing higher expression of JMJD3 in IPF-derived AT2 cells by pseudobulk re-analysis of Habermann et al dataset compared to non-diseased control. Similarly, JMJD3 has higher expression in AT2 cells from older donors in our dataset. Significance testing by two-tailed Wilcoxon rank sum test for single cell data, and one-tailed for pseudobulk data. (G.) IL1β stimulation of AT2 cells leads to expression of NF-kB markers such as TNFAIP3 , chemokines such as CCL2 , and histone demethylase JMJD3 by qPCR. (H.) IL1β-pre-treated AT2 cells have higher expression of KRT5 , KRT17 , and TP63 in 14-day organoid co-culture with primary fibroblasts by qPCR compared to untreated control. (I.) IL1β-pre-treated AT2 cells have higher expression of KRT5 but not KRT17 compared to untreated control after 7 days of culture in mesenchyme-free conditions with roxadustat. This effect is abrogated if IL1β pre-treatment is performed with JMJD3 inhibitor GSKJ4. (J.) Immunofluorescence staining of cytospins confirming increased KRT5+/KRT17+ basal cells with IL1β pre-treatment of AT2 cells followed by 7 days of mesenchyme-free culture with roxadustat. Both untreated control and IL1β/GSKJ4 pre-treated AT2 cells have <1% KRT5+/KRT17+ basal cells by immunostaining after roxadustat for 7 days, while the number of KRT5+/KRT17+ basal cells is around 5-10% in IL1β pre-treated cells, pointing to a subset of AT2 cells being IL1β-responsive. Statistical significance was determined by unpaired t-test (F) , paired t-test (G, H) , and ordinary one-way ANOVA (I) .

Article Snippet: When applicable, cells were treated with IL-1β (25ng/ml, HY-P7028, Med Chem Express), GSKJ4, a JMJD3 inhibitor, (5-10μM, HY-15648B, Med ChemExpress), BAY 11-7082, an NF-KB inhibitor, (10 –25 μM, Cat# 196870, Sigma), roxadustat, a prolyl hydroxylase inhibitor and HIF agonist (15 μM, FG-4592, MedChemExpress), TGFβ1 (2ng/mL, Cat#100B, R&D Systems), or LATS inhibitor (LATSI or Lats-IN-1, 10uM, HY-138489, MedChemExpress).

Techniques: Single Cell, Derivative Assay, Activity Assay, In Vitro, Isolation, Co-Culture Assay, Expressing, Control, Two Tailed Test, One-tailed Test, Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Epigenetic de-repression of basal cell metaplasia in aging AT2 cells is a risk factor for idiopathic pulmonary fibrosis (IPF)

doi: 10.64898/2026.06.09.731212

Figure Lengend Snippet: (A) Schematic of experimental setup for CUT&Tag profiling of KLF5 binding sites. AT2 cells from old donors were treated with LATS inhibitor to induce AT1 marker expression via YAP/TAZ activation for 3 days, a portion of cells harvested for CUT&Tag, and the rest cultured in the presence of HIF agonist roxadustat for 2 days to drive acquisition of basal cell markers, prior to being collected for repeat CUT&Tag. Results were compared with CUT&Tag for KLF5 in NGFR+ basal cells from an IPF donor. (B) Representative genomic tracks of normalized read depth from CUT&Tag sequencing for KLF5 in AT2 cells treated with LATS inhibitor for 3 days to drive an AT1-like cell state (track 1, “AT2 + LATSI”), treated with LATS inhibitor for 3 days and then roxadustat for 2 days (track 2, “AT2 + LATSI -> ROX”), and finally IPF-derived basal cells (track 3, “IPF BC”). Select marker loci ( AGER as AT1 cell marker, KRT5 as a basal cell marker) are shown. Peak height represents stronger KLF5 binding, while −log10(q) values shown represent the average statistical significance of relevant peak calls in the promoter region. There is strong binding to AGER promoter with LATS inhibition that decreases with HIF agonism, while KRT5 promoter binding increases. Conversely, there is strong binding to KRT5 promoter in IPF-derived basal cells and no significant binding to AGER promoter. Of note, KLF5 also binds the KRT17 promoter (not shown) in both roxadustat treated and IPF basal cells. (C) Enrichment around transcription start site (TSS) for KLF5 in differentially bound genes between LATS-inhibitor treated cells and IPF-derived basal cells. Genes with higher promoter binding in each condition were labeled “AT1 genes” and “BC genes”, respectively. (D) Identification of shared genes with any KLF5 binding (peak q-value cutoff < 0.01) within their promoter region (defined as TSS +/− 3kb) across conditions, irrespective of peak height (i.e. binding intensity). After treatment with roxadustat, a greater proportion of KLF5-bound genes are shared with IPF basal cells in LATS-treated AT1-like cells (increase from 77% to 81%). (E) Schematic of experimental setup for basaloid cell fate induction in mesenchyme-free conditions in old AT2 cells. (F) Activation of KRT17 by TGFβ1 is JMJD3-dependent and is specific for the basaloid cell fate (KRT5- /KRT17+). (G) Expression of KRT17 does not depend on AT2 cell priming or age, as shown by robust mRNA expression in young AT2 cells by day 4 of TGFβ1 treatment. (H) Immunoprecipitation of KLF5 and Western Blot for SMAD2/3, HIF1α, and YAP from IPF-derived basal cells treated with either roxadustat or TGFβ1. HIF1α stabilization with roxadustat drives its association with KLF5 while also diminishing KLF5-SMAD2/3 association, which instead is strongest in the presence of TGFβ1. Similarly, HIF1α stabilization also drives association of KLF5 with YAP, a key driver of AT2-to-AT1 transdifferentiation. Statistical significance was determined by Poisson local test with BH correction as in (A) , by ordinary one-way ANOVA (F) and two-way ANOVA (G) .

Article Snippet: When applicable, cells were treated with IL-1β (25ng/ml, HY-P7028, Med Chem Express), GSKJ4, a JMJD3 inhibitor, (5-10μM, HY-15648B, Med ChemExpress), BAY 11-7082, an NF-KB inhibitor, (10 –25 μM, Cat# 196870, Sigma), roxadustat, a prolyl hydroxylase inhibitor and HIF agonist (15 μM, FG-4592, MedChemExpress), TGFβ1 (2ng/mL, Cat#100B, R&D Systems), or LATS inhibitor (LATSI or Lats-IN-1, 10uM, HY-138489, MedChemExpress).

Techniques: Binding Assay, Marker, Expressing, Activation Assay, Cell Culture, Sequencing, Derivative Assay, Inhibition, Labeling, Immunoprecipitation, Western Blot

Journal: Toxicology and applied pharmacology

Article Title: Nickel exposure disrupts epigenetic repression of developmental genes in mouse embryonic stem cells

doi: 10.1016/j.taap.2026.117810

Figure Lengend Snippet: Cell differentiation associated pathways are enriched among the genes differentially expressed by Ni exposure. A. E14Tg2a mESCs were treated with three concentrations of NiCl 2 (100, 150 and 200 μM) for two exposure durations (4 or 6 days). Differentially expressed (DE) genes were identified using RNA-Seq analysis with DESeq2 (|log 2 FC| ≥ log 2 (1.5) and adjusted p -value <0.05 and RPKM ≥1 in at least one condition) by comparing Ni-treated with time-matched untreated controls. RNA-seq results are displayed as volcano plots. Red circles indicate upregulated genes, blue circles indicate downregulated genes, and black circles indicate genes that were not significantly differentially expressed. B. Overrepresentation analysis (ORA) of DE genes was performed to identify enriched Gene Ontology (GO) biological process terms. The top 10 enriched GO terms for each treatment condition are shown for 4-day treatments (top row) and 6-day treatments (bottom row). The y -axis lists the top 10 GO terms, and the x -axis shows the gene ratio (proportion of DE genes annotated to each term). Circle color indicates the number of DE genes associated with each term, and circle size indicates the adjusted p -value (corrected for multiple testing with Benjamini-Hochberg correction). The top 10 GO terms were selected based on the lowest adjusted p -values and ranked by gene ratio. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For demethylase inhibitor experiments, E14Tg2a cells were treated with 150 μM NiCl 2 and 3 μM GSK-J4 (HY-15648B, MedChemExpress) for 4 days.

Techniques: Cell Differentiation, RNA Sequencing

Journal: Toxicology and applied pharmacology

Article Title: Nickel exposure disrupts epigenetic repression of developmental genes in mouse embryonic stem cells

doi: 10.1016/j.taap.2026.117810

Figure Lengend Snippet: Ni exposure induces genes associated with mesoderm and endoderm developmental programs. A. Heatmaps display log 2 fold-change values for genes upregulated across all three NiCl 2 treatment conditions (100, 150, and 200 μM) and annotated with the GO biological process term “cell differentiation” (GO:0030154). Genes were ranked by their average log 2 fold-change across treatment conditions, and the top 10 genes are shown for day 4 (left) and day 6 (right). Color intensity represents log 2 fold-change. B. Heatmaps of log 2 fold-change values for genes annotated with the GO biological process terms “mesoderm development” (GO:0007498, left), “endoderm development” (GO:0007492, middle), and “ectoderm development” (GO:0007398, right). Each heatmap displays expression changes across three NiCl 2 concentrations (100, 150, and 200 μM) at day 4 and day 6. Genes are ordered by their average log 2 fold-change across treatment conditions. Color intensity represents log 2 fold-change. C. Representative images of E14Tg2a mESC colonies following 4- and 6-day exposure to increasing concentrations of NiCl 2 .

Article Snippet: For demethylase inhibitor experiments, E14Tg2a cells were treated with 150 μM NiCl 2 and 3 μM GSK-J4 (HY-15648B, MedChemExpress) for 4 days.

Techniques: Cell Differentiation, Expressing

Journal: Toxicology and applied pharmacology

Article Title: Nickel exposure disrupts epigenetic repression of developmental genes in mouse embryonic stem cells

doi: 10.1016/j.taap.2026.117810

Figure Lengend Snippet: Polycomb Repressive Complex 2 (PRC2) components are enriched among predicted transcriptional regulators of Ni-upregulated genes. BART analysis of genes upregulated (A, B) and downregulated (C, D) by Ni exposure. (A, C) Predicted transcriptional regulators were ranked based on Irwin–Hall p -values across all NiCl 2 concentrations (100, 150, and 200 μM) and exposure durations (4 and 6 days). The average rank across conditions was used to identify the top 10 regulators, shown for day 4 (left) and day 6 (right) for upregulated (A) and downregulated (C) genes. (B, D) Area under the curve (AUC) values from ChIP-seq enrichment analyses for the top 10 experiments with the highest average rank across all NiCl 2 treatment conditions (100, 150, and 200 μM) and exposure durations (4 and 6 days). Results are shown for day 4 (left) and day 6 (right) for upregulated (B) and downregulated (D) genes. ChIP-seq datasets generated in mESCs are indicated with an asterisk (*) and those from mESCs differentiated toward the mesoderm lineage are indicated with a double asterisk (**).

Article Snippet: For demethylase inhibitor experiments, E14Tg2a cells were treated with 150 μM NiCl 2 and 3 μM GSK-J4 (HY-15648B, MedChemExpress) for 4 days.

Techniques: ChIP-sequencing, Generated

Journal: Toxicology and applied pharmacology

Article Title: Nickel exposure disrupts epigenetic repression of developmental genes in mouse embryonic stem cells

doi: 10.1016/j.taap.2026.117810

Figure Lengend Snippet: Ni-induced gene activation is associated with bivalent genes and reduced H3K27me3 levels. A. Distribution of promoter classes among upregulated genes across NiCl 2 treatment conditions. Bars show the proportion of upregulated genes belonging to four promoter classes in mESCs: K4+K27 (bivalent; yellow), K4-only (green), K27-only (red), and unmarked (blue), where K4 and K27 denote H3K4me3 and H3K27me3, respectively. Promoter class annotations in mESCs were obtained from Liu et al. . The bar labeled ‘background’ shows genome-wide proportions of the promoter classes. Enrichment of bivalent promoters among upregulated genes was evaluated using Fisher’s exact test (one- versus -all comparison), comparing bivalent genes to all other promoter classes in the upregulated set relative to the genome-wide background ( p < 0.0001****; ns, not significant). B. Gene set enrichment analysis (GSEA) showing enrichment of a bivalent gene set in cells treated with 150 μM NiCl 2 for 4 days compared with time-matched, untreated controls. The bivalent gene set consisting of genes annotated as K4+K27 in mESCs was obtained from Liu et al. . C. ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment at mesodermal gene promoters in mESCs treated with 0 or 150 μM NiCl 2 for 4 days. Data are shown as fold enrichment relative to IgG. Error bars represent SD of three biological replicates. Statistical significance was determined using a t -test ( p < 0.05*; p < 0.01**; ns, not significant). D. Western blot analysis showing levels of H3K4me3 and H3K27me3 in mESCs treated with 0 or 150 μM NiCl 2 for 4 days. Histone H3 was used as a loading control. Representative blots are shown in the upper panel, with quantification of band intensities (normalized to H3 and shown as fold change relative to control) in the lower panels. Error bars represent SD of three biological replicates. Statistical significance was assessed using a t -test (ns, not significant). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For demethylase inhibitor experiments, E14Tg2a cells were treated with 150 μM NiCl 2 and 3 μM GSK-J4 (HY-15648B, MedChemExpress) for 4 days.

Techniques: Activation Assay, Labeling, Genome Wide, Comparison, ChIP-qPCR, Western Blot, Control

Journal: Toxicology and applied pharmacology

Article Title: Nickel exposure disrupts epigenetic repression of developmental genes in mouse embryonic stem cells

doi: 10.1016/j.taap.2026.117810

Figure Lengend Snippet: H3K27me3 demethylase inhibitor prevents Ni-induced activation of mesoderm-associated genes. A. RT-qPCR analysis of mesodermal gene expression in mESCs exposed to 150 μM NiCl 2 for 4 days, with or without KDM6A/B-selective inhibitor GSK-J4 (3 μM). Actb was used as an internal control. Error bars represent SD of three biological replicates. Statistical significance was determined using a t -test ( p < 0.05*; p < 0.01**; p < 0.001***). B. ChIP-qPCR analysis of H3K27me3 enrichment at mesodermal gene promoters in mESCs treated with 150 μM NiCl 2 for 4 days, with or without KDM6A/B inhibitor GSK-J4 (3 μM). Data are shown as fold enrichment relative to IgG. Error bars represent SD of three biological replicates. Statistical significance was determined using a t -test ( p < 0.01**; p < 0.001***; ns, not significant). C. Representative images of mESC colonies following 4-day exposure to 150 μM NiCl 2 , with or without the KDM6A/B inhibitor GSK-J4 (3 μM).

Article Snippet: For demethylase inhibitor experiments, E14Tg2a cells were treated with 150 μM NiCl 2 and 3 μM GSK-J4 (HY-15648B, MedChemExpress) for 4 days.

Techniques: Activation Assay, Quantitative RT-PCR, Gene Expression, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Heatmaps showing CTCF and H3K27me3 signal intensity at CTCF binding sites that demarcate transitions in H3K27me3-enriched heterochromatin domains (L2FC H3K27me3 > 1). ( B ) Kacme ChIP-seq enrichment centered at CTCF-H3K27me3 transition sites , clustered by Kacme levels (C1-C4). Upper and lower plots show Kacme enrichment profiles associated with transitions out of (upper) or into (lower) H3K27me3 domains. Purple arrowheads denote the center of the CTCF-anchored transition. ( C ) BRD2 ChIP-seq signal centered on CTCF sites for the four CTCF-H3K27me3 transition clusters defined in . Transitions enriched for Kacme (C1) show the strongest co-enrichment for BRD2. ( D ) Representative western blot of global Kacme levels following 72 h of GSK-J4 treatment (2.5 μM). ( E ) Metaplots of spike-in normalized H3K27me3 CUT&RUN signal in DMSO-treated HEK293T cells and HEK293T cells treated with 5 μM GSK-J4 for 6 h or 24 h. Inhibition of KDM6A/B results in a global increase in gene body H3K27me3 levels. ( F ) Genome browser tracks showing Kacme profiles in control HEK293T cells and H3K27me3 profiles in control, GSK-J4 (6 h), or GSK-J4 (24 h)-treated HEK293T cells. GSK-J4 treatment leads to increased H3K27me3 levels; however, H3K27me3 spreading does not extend beyond the Kacme-marked boundary into the SFSWAP locus. ( G ) L2FC in spike-in normalized H3K27me3 levels (24 h GSK-J4 vs. control) centered on CTCF transition sites (C1 and C4, as defined in ). Purple arrowheads denote the center of the CTCF-anchored transition. ( H ) MA plot displaying L2FC in spike-in normalized RNA expression levels (24 h GSK-J4 vs. control) versus mean expression. Red points indicate significantly upregulated genes (L2FC > 1, FDR-adjusted p < 0.05) and significantly downregulated genes (L2FC < −1, FDR-adjusted p < 0.05). ( I ) Box plots showing the L2FC in RNA expression (24 h GSK-J4 vs. control) at genes proximal to CTCF transition sites (within 10 kb of C1-C4 transition sites, as defined in ). Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-hoc test. * = p < 0.05.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Inhibition, Control, RNA Expression, Expressing

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A) Heatmaps showing H3K27me3 signal intensity in untreated HEK293T cells at CTCF binding sites that demarcate transitions in H3K27me3-enriched heterochromatin domains (L2FC H3K27me3 > 1). Transition sites were stratified by Kacme co-enrichment levels (C1-C4, see ). Transition sites with high Kacme levels (C1) exhibit reduced H3K27me3 spreading into neighboring euchromatin regions. (B) Representative western blot of global H3K27me3 levels following 24 h of GSK-J4 treatment (0-10 μM). (C) Representative western blot of global H3K27me3 levels following 6 or 24 h of GSK-J4 treatment (5 μM). (D) Metaplots of spike-in normalized H3K27me3 CUT&RUN signal in DMSO-treated HEK293T cells and HEK293T cells treated with 5 μM GSK-J4 for 6 h or 24 h. Inhibition of KDM6A/B results in a global increase in promoter H3K27me3 levels. (E) Principal component analysis (PCA) plot of spike-in normalized TimeLapse-seq read counts, transformed by the rlog function in DESeq2. DMSO- and 24 h GSK-J4-treated samples clustered separately.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Binding Assay, Western Blot, Inhibition, Transformation Assay

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Relative promoter accessibility (±50 bp of the TSS) in HEK293T cells as determined by ATAC-seq. Normalized ATAC-seq signal was binned by promoter Kacme levels (±1 kb of the TSS) as determined by ChIP-seq. ( B ) Genome browser visualization of ATAC-seq and Kacme ChIP-seq profiles at selected loci on chromosome 19 in HEK293T cells. ( C ) Heatmaps of Kacme ChIP-seq and H3K27me3 CUT&RUN signal centered on transcription start sites (±2.5 kb) across all annotated HEK293T promoter regions. ( D ) Relative promoter accessibility in HEK293T cells for genes in the 25 th or 75 th percentiles of Kacme CUT&RUN levels. Data are binned by H4K5ac CUT&RUN signal. ( E ) Left: Representative western blot of histones from HEK293T cells treated with the transcription inhibitors triptolide (10 μM, 1 h) or flavopiridol (500 nM, 1 h). Right: Venn diagram showing the overlap between genes with a promoter-proximal Kacme peak (±3 kb of the TSS) in untreated HEK293T cells (light blue) and in triptolide-treated HEK293T cells (light red). Kacme peaks were identified using MACS2. ( F ) Genome browser tracks showing ATAC-seq, Kacme, H3K4me1, H3K27ac, and H3K4me3 signal profiles across an enhancer-rich locus in HEK293T cells. ( G ) Heatmaps and aggregated signal profiles for ATAC-seq, Kacme, H3K27ac, H3K4me1, and H3K27me3 across six enhancer clusters identified in HEK293T cells by k-means clustering. Aggregated profiles show the mean signal across enhancer regions within each cluster. ( H ) HOMER known motif analysis for enhancer clusters 1 and 2 , showing the top 20 enriched motifs with their best-match transcription factors and associated p-values. FOX proteins highlighted in magenta, CTCF highlighted in green. ( A, D ) Distribution means compared with two-tailed unpaired Wilcoxon test for two biological replicates. *** = p < 0.001.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: ChIP-sequencing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Relative promoter accessibility in THP-1 cells for genes in the 25 th or 75 th percentiles of Kacme ChIP-seq levels. Data are binned by H4K5ac ChIP-seq signal. ( B ) Relative promoter accessibility in THP-1 cells for genes in the 25 th or 75 th percentiles of Kacme ChIP-seq levels. Data are binned by H3K27ac ChIP-seq signal. ( C ) Genome browser visualization of Kacme ChIP-seq (untreated) and Kacme CUT&RUN (triptolide-treated) profiles at representative loci on chromosome 5 in HEK293T cells. ( B-C ) Distribution means compared with two-tailed unpaired Wilcoxon test for two biological replicates. *** = p < 0.001; * = p < 0.05; NS = Not Significant.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: ChIP-sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A-B) Genome browser tracks showing Kacme ChIP-seq and ATAC-seq signal profiles at representative TSS-distal regions (highlighted in blue) in (A) HEK293T and (B) THP-1 cells. (C-D) Genomic feature distribution of consensus Kacme ChIP-seq peaks in (C) HEK293T and (D) THP-1 cells. Peaks were annotated using the ChIPseeker Bioconductor package. (E) Heatmap showing HEK293T Kacme ChIP-seq fold enrichment across HEK293T-specific enhancer regions (± 1kb from enhancer boundaries). (F) Overlap analysis between Kacme peaks (called with MACS2) and ChromHMM chromatin state annotations from the Kellis laboratory 18-state model in K562 cells (ENCSR961HFL, ). Bars indicate the percentage of base pairs within each ChromHMM state that overlap Kacme peak regions. (G) Example genome browser tracks showing H3K4me1, Kacme, and H3K27ac ChIP-seq enrichment at annotated enhancer regions in HEK293T cells (highlighted in blue). (H) Profile plot showing S2 Kacme ChIP-seq enrichment across annotated S2 enhancer regions (± 2kb from enhancer boundaries). (I) Example genome browser track showing ATAC-seq, H3K4me1, Kacme, and H3K27ac co-occupancy at an Act5c -proximal enhancer region in Drosophila S2 cells (highlighted in blue). Notably, H3K4me3 signal is absent, consistent with the enhancer identity of this region rather than a promoter-associated state. (J) Profile plots of Kacme ChIP-seq enrichment and ATAC-seq signal across annotated K562 silencer elements (± 1kb), obtained from . Silencer elements were clustered by ATAC-seq signal. (K) Example genome browser tracks showing Kacme and ATAC-seq signal profiles at annotated silencer elements in K562 cells (highlighted in red). (L) Metaplot analysis of average CpG methylation levels centered on the top 5,000 Kacme or H3K27ac ChIP-seq peak summits (ranked by their respective enrichment scores) in HEK293T cells. (M) Metaplot analysis of average CpG methylation levels centered on the top 2,000 peaks ranked by relative H3K27ac-to-Kacme signal or Kacme-to-H3K27ac signal in HEK293T cells.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: ChIP-sequencing, CpG Methylation Assay

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Histone mark and transcription factor emission probabilities, along with chromatin state definitions, for the 15-state ChromHMM model generated in wild-type K562 cells. Consensus peak BED files for histone PTMs and transcription factors in K562 cells were used as input for ChromHMM. Features were hierarchically clustered using Euclidean distance and complete-linkage clustering, while chromatin states were displayed in numerical order. ( B ) Genomic annotation enrichment for ChromHMM State 12, showing relative overlap with gene features and CpG islands. ( C ) Metaplot analysis of average CpG methylation levels centered on consensus Kacme peak summits in HEK293T cells. LOESS-smoothed curves with 95% confidence intervals show the mean signal ± CI across 100-bp bins spanning ± 20kb around the peak center for each biological replicate. ( D ) HOMER known motif analysis for ChromHMM State 12 genomic regions. The top 10 enriched motifs are ranked by significance with their best-match transcription factors and associated p-values. ( E ) Genome browser tracks showing Kacme ChIP-seq signal at the MAFB and LPP loci in THP-1 cells treated with DMSO or PMA. Blue bars denote macrophage-specific enhancer regions, as defined by EnhancerAtlas 2.0. ( F ) Left: MA plot depicting the changes in promoter Kacme ChIP-seq signal in THP-1 cells following PMA treatment. Promoters with significantly differential Kacme levels are indicated in red (L2FC > 0, FDR-adjusted p < 0.05) and blue (L2FC < 0, FDR-adjusted p < 0.05). Right: ARCHS4 Tissues enrichment analysis for genes with significantly upregulated promoter Kacme signal (n = 2,400 genes). ( G ) Normalized ATAC-seq signal changes following PMA treatment in THP-1 cells, plotted over significantly upregulated Kacme peaks (PMA/DMSO) located within 3 kb of gene TSSs. ( H ) Left: Scatterplot comparing the changes in promoter Kacme enrichment and RNA-seq levels following PMA treatment in THP-1 cells. Pearson correlation analysis was performed to assess the changes in L2FC values across experimental datasets. Right: Boxplots comparing changes in promoter Kacme levels at genes downregulated (L2FC < −1, FDR-adjusted p < 0.05), unchanged, or upregulated (L2FC > 1, FDR-adjusted p < 0.05) by RNA-seq following PMA treatment. Statistical significance between groups was determined using pairwise Wilcoxon rank-sum tests. *** = p < 0.001. ( I ) Genome browser tracks of Kacme, H3K27ac, H3K27me3, and H3K9me3 ChIP-seq profiles in THP-1 monocytes versus macrophages at the NOL4L locus.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Generated, CpG Methylation Assay, ChIP-sequencing, RNA Sequencing

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Chromosome spreads were prepared from HeLa cells arrested in nocodazole and immunostained with antibodies against Kacme (green) and centromeres using anti-centromere antibody (ACA, magenta). DNA was counterstained with DAPI (blue). Insets show representative chromosome regions with Kacme retained on condensed mitotic chromatin. Scale bar, 5 μm. ( B ) Protocol to synchronize HEK293T cells and arrest at the G2/M boundary using a nocodazole block/mitotic shake-off. Matched CUT&RUN (against Kacme and H4K5ac), ATAC-seq, and nascent RNA sequencing were performed at several timepoints across the M/G1 transition. ( C ) Genome browser tracks showing a representative gene, ATF4 , that is marked by Kacme throughout the mitosis-G1 phase transition. ( D ) Heatmap showing hierarchical k-means clustering of normalized Kacme CUT&RUN signal across promoter regions during mitotic release. Four major clusters (C1-C4) were identified, with 1,579 genes in C4 displaying the strongest Kacme signal during mitosis. Increasing signal intensity is indicated by a red color scale. ( E ) Transcription factor enrichment analysis of genes associated with Cluster 4 . Top enriched motifs include mitotically retained TFs such as MYC, NFYA, and POLR2A. ( F ) Differential binding analysis of regions of enrichment from Kacme and H4K5ac CUT&RUN in mitotically arrested cells (T0). Black points are regions of significant difference (p-value < 0.05). ( G ) Normalized ATAC-seq signal at the transcription start sites of genes associated with each Kacme cluster (C1-C4; ) across selected mitotic release time points (T0, T40, T105, T300). Promoters with the highest level of mitotic Kacme enrichment showed the most significant gain in accessibility upon release from mitosis into early-G1. ( H ) Heatmap showing nascent transcription levels (TimeLapse-seq) for genes that first reach ≥0.5-fold above asynchronous levels during mitotic release. Within each timepoint (columns), genes are rank-ordered based on their transcription levels. Nascent transcription values are shown as Z-scores of RPKM. ( I ) Right: Median RPKM of nascent transcription across mitotic release for genes in each Kacme cluster (C1-C4; ). Left: Median RPKM of nascent transcription across mitotic release for genes grouped by mitotic H4K5ac levels (C1-C4). ( J ) Comparison of nascent transcriptional dynamics at genes preferentially marked by Kacme, H4K5ac, or both, during the mitotic release time course. Median RPKM values of nascent transcription for genes associated with each set are shown across mitotic release timepoints.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Blocking Assay, RNA Sequencing, Sublimation, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A) Western blot analysis of H4Kacme, H4K5ac, and H3S10P in asynchronous HEK293T cells and HEK293T cells collected at various timepoints following release from nocodazole-mediated mitotic arrest. (B) Flow cytometry analysis of DNA content (propidium iodide) in asynchronous HEK293T cells and HEK293T cells collected at various timepoints following release from nocodazole-mediated mitotic arrest. (C) Heatmaps of normalized Kacme CUT&RUN signal at HEK293T-specific enhancer regions throughout the mitotic-release time course. (D) Genome browser tracks showing H3K4me3 in asynchronous cells and mitotic-Kacme/H4K5ac CUT&RUN signal at the TSC22D3 enhancer region (shaded in grey). (E) Differential binding analysis of regions of enrichment from Kacme CUT&RUN performed in mitotically arrested cells (T0) and asynchronous cells. Black points are regions of significant difference (|L2FC| > 1, p-value < 0.05). (F) Representative genome browser tracks showing CTCF enrichment in asynchronous HEK293T cells and Kacme CUT&RUN signal in mitotically arrested (T0) HEK293T cells. FIMO-predicted CTCF binding sites are annotated in magenta. (G) Aggregate Kacme CUT&RUN signal in mitotically arrested (T0) HEK293T cells at forward and reverse-oriented FIMO-predicted CTCF binding sites. (H) Biological pathway enrichment analysis for genes with significantly high relative promoter Kacme-to-H4K5ac ratios in mitotically arrested (T0) HEK293T cells. (I) Genome browser view of H4K5ac and Kacme CUT&RUN signal across the HIST1 locus during the mitotic-release time course. (J) Metaplots of H4K5ac and Kacme CUT&RUN signal at promoter regions (±3 kb around TSS) across the mitotic-release time course. (K) Metaplots of H4K5ac and Kacme CUT&RUN signal at HEK293T-specific enhancer regions across the mitotic-release time course.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Western Blot, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A) Normalized ATAC-seq signal at four timepoints following mitotic release, centered on transcription start sites (±3 kb) across all annotated promoter regions. (B) Boxplots showing promoter ATAC-seq signal (±3 kb of TSS) in mitotically arrested (T0) HEK293T cells. Genes are grouped into four clusters (C1-C4) defined by mitotic Kacme levels (see ). Increasing Kacme is associated with higher chromatin accessibility in mitotically arrested cells. Statistical significance was determined using pairwise Wilcoxon rank-sum tests. *** = p < 0.001. (C) Mean nascent transcription (RPKM) across the mitotic-release time course, as assayed by TimeLapse-seq. Nascent transcription globally increases following mitotic exit, consistent with broad transcriptional reactivation. (D) Median RPKM values of nascent transcription for genes associated with each Kacme cluster (C1-C4; ) across mitotic release timepoints. Values were further scaled to reflect transcriptional reactivation relative to T40 and asynchronous levels, enabling direct comparison of reactivation dynamics across clusters. (E) Comparison of nascent transcriptional dynamics at genes preferentially marked by Kacme, H4K5ac, or both, during mitosis (T0). Differential binding analysis was used to identify promoters with significantly differential Kacme and H4K5ac enrichment in mitotically arrested cells (T0), as described in . Median RPKM values of nascent transcription for genes associated with each set are shown across mitotic release timepoints. (F) Pathway enrichment analysis for genes with high relative mitotic Kacme-to-H4K5ac ratios (see ), that exhibit early reactivation following mitotic release. Genes were classified as early reactivation genes if they first reached nascent transcription levels ≥0.5-fold above asynchronous levels within 40 minutes of mitotic release. (G) Pathway enrichment analysis for genes with high relative mitotic Kacme-to-H4K5ac ratios (see ), that exhibit late reactivation following mitotic release. Genes were classified as late reactivation genes if they first reached nascent transcription levels ≥0.5-fold above asynchronous levels 300 minutes after mitotic release.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Comparison, Binding Assay

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Genome browser visualization of bulk H3K27me3, Kacme, H4Kac, and H3K27ac profiles at the X chromosome XIST locus in female-derived HEK293T cells. ( B ) Genome browser visualization of bulk H3K27me3, Kacme, H4Kac, and H3K27ac profiles at the escapee genes DDX3X , TBL1X , and KDM5C in female-derived HEK293T cells. ( C ) Genome browser visualization of bulk Kacme ChIP-seq signal at the XIST locus in male-derived THP-1 cells. ( D ) Distribution of log-transformed cumulative Kacme ChIP-seq signal across the gene bodies of X escapee, X inactivated, and autosomal genes. Dashed lines represent the maxima of the distribution for each category of genes. ( E ) Top: Bulk Kacme ChIP-seq enrichment across the XIST locus in untreated HEK293T cells. Bottom: Allelic contribution of Kacme ChIP-seq and RNA-seq reads mapping to the Xa (active X, blue) and Xi (inactive X, red) at XIST SNP1 and SNP2 positions. ( F ) Top: Bulk Kacme ChIP-seq enrichment across the DDX3X locus in untreated HEK293T cells. Bottom: Allelic contribution of Kacme ChIP-seq reads mapping to the reference (green) and alternative (grey) alleles at two DDX3X SNPs.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Derivative Assay, ChIP-sequencing, Transformation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A) Genome browser views showing HEK293T H3K27me3 CUT&RUN and Kacme, H3K27ac, and H4Kac ChIP-seq profiles at the borders of representative X chromosome heterochromatin domains. (B) Metagene analysis of HEK293T Kacme ChIP-seq enrichment at the 5’ and 3’ boundaries of broad H3K27me3 domains (n = 4,162). (C) Genome browser views showing H3K27me3 CUT&RUN and Kacme and H3K27ac ChIP-seq profiles at the border of a heterochromatin domain proximal to the IGF2R locus. (D) Left: Scatter plot comparing overall Kacme and H3K27ac ChIP-seq fold enrichment in HEK293T cells, measured within ±1 kb of n = 4,162 heterochromatin boundaries. Pearson correlation values and significance are indicated. Right: Boxplot showing the log fold difference in Kacme and H3K27ac enrichment at boundary-proximal regions. (E) Metaplots of Kacme ChIP-seq fold enrichment centered on forward- and reverse-oriented FIMO-predicted CTCF binding sites (± 1kb). (F) Heatmaps of Kacme CUT&RUN signal centered on forward- and reverse-oriented FIMO-predicted CTCF binding sites (± 2kb). CUT&RUN from two biological replicates are shown. (G) Metaplots of H3K27ac ChIP-seq fold enrichment centered on forward- and reverse-oriented FIMO-predicted CTCF binding sites (± 1kb). (H) Violin plots showing fold change in THP-1 promoter Kacme ChIP-seq signal (PMA/DMSO), binned by quartiles of changes in Hi-C loop strength (as reported in ). Genes exhibiting increased PMA-induced looping (Q4) are associated with increased Kacme signal, while genes exhibiting decreased looping (Q1) are associated with decreased Kacme. Statistical significance was determined using pairwise Wilcoxon rank-sum tests. *** = p < 0.001.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: ChIP-sequencing, Binding Assay, Hi-C

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Metagene analysis of HEK293T Kacme CUT&RUN signal across broad H3K27me3 domains showing enrichment near domain boundaries. ( B ) Genome browser view of an example locus in HEK293T cells with Kacme enrichment at the border of a broad H3K27me3 heterochromatin domain. ( C ) Differential binding analysis of regions of enrichment from Kacme and H3K27ac ChIP-seq in untreated HEK293T cells. Black points are regions of significant difference (FDR-adjusted p < 0.05). ( D ) HOMER known motif analysis for regions with significantly high relative Kacme-to-H3K27ac ratios . The top two enriched motifs correspond to CTCF and CTCFL. ( E ) Genome browser view of the AMOT locus in HEK293T cells, which shows co-enrichment for Kacme and CTCF at the border of a broad H3K27me3 heterochromatin domain. ( F ) Metaplots of Kacme ChIP-seq and CTCF CUT&RUN signal centered on forward- and reverse-oriented FIMO-predicted CTCF binding sites (± 1kb). Sites are stratified by Kacme enrichment levels. ( G ) Genome tracks from DMSO- and PMA-treated THP-1 cells showing differences in Kacme, CTCF, H3K27me3, and H3K9me3 profiles at the CLIP2 locus. The highlighted region marks a site that loses both Kacme and CTCF binding upon PMA-induced differentiation and exhibits spreading of heterochromatin. ( H ) Violin plots illustrating the L2FC in promoter Kacme ChIP-seq signal at genes that either remain static or exhibit significant increases in Hi-C chromatin looping upon PMA-induced differentiation of THP-1 cells (as reported in ). Statistical significance was determined using pairwise Wilcoxon rank-sum tests. *** = p < 0.001.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Binding Assay, ChIP-sequencing, Hi-C

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: ( A ) Heatmaps showing CTCF and H3K27me3 signal intensity at CTCF binding sites that demarcate transitions in H3K27me3-enriched heterochromatin domains (L2FC H3K27me3 > 1). ( B ) Kacme ChIP-seq enrichment centered at CTCF-H3K27me3 transition sites , clustered by Kacme levels (C1-C4). Upper and lower plots show Kacme enrichment profiles associated with transitions out of (upper) or into (lower) H3K27me3 domains. Purple arrowheads denote the center of the CTCF-anchored transition. ( C ) BRD2 ChIP-seq signal centered on CTCF sites for the four CTCF-H3K27me3 transition clusters defined in . Transitions enriched for Kacme (C1) show the strongest co-enrichment for BRD2. ( D ) Representative western blot of global Kacme levels following 72 h of GSK-J4 treatment (2.5 μM). ( E ) Metaplots of spike-in normalized H3K27me3 CUT&RUN signal in DMSO-treated HEK293T cells and HEK293T cells treated with 5 μM GSK-J4 for 6 h or 24 h. Inhibition of KDM6A/B results in a global increase in gene body H3K27me3 levels. ( F ) Genome browser tracks showing Kacme profiles in control HEK293T cells and H3K27me3 profiles in control, GSK-J4 (6 h), or GSK-J4 (24 h)-treated HEK293T cells. GSK-J4 treatment leads to increased H3K27me3 levels; however, H3K27me3 spreading does not extend beyond the Kacme-marked boundary into the SFSWAP locus. ( G ) L2FC in spike-in normalized H3K27me3 levels (24 h GSK-J4 vs. control) centered on CTCF transition sites (C1 and C4, as defined in ). Purple arrowheads denote the center of the CTCF-anchored transition. ( H ) MA plot displaying L2FC in spike-in normalized RNA expression levels (24 h GSK-J4 vs. control) versus mean expression. Red points indicate significantly upregulated genes (L2FC > 1, FDR-adjusted p < 0.05) and significantly downregulated genes (L2FC < −1, FDR-adjusted p < 0.05). ( I ) Box plots showing the L2FC in RNA expression (24 h GSK-J4 vs. control) at genes proximal to CTCF transition sites (within 10 kb of C1-C4 transition sites, as defined in ). Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-hoc test. * = p < 0.05.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Inhibition, Control, RNA Expression, Expressing

Journal: bioRxiv

Article Title: Histone H4 acetyl-methyllysine marks accessible chromatin that resists compaction

doi: 10.64898/2026.04.17.718779

Figure Lengend Snippet: (A) Heatmaps showing H3K27me3 signal intensity in untreated HEK293T cells at CTCF binding sites that demarcate transitions in H3K27me3-enriched heterochromatin domains (L2FC H3K27me3 > 1). Transition sites were stratified by Kacme co-enrichment levels (C1-C4, see ). Transition sites with high Kacme levels (C1) exhibit reduced H3K27me3 spreading into neighboring euchromatin regions. (B) Representative western blot of global H3K27me3 levels following 24 h of GSK-J4 treatment (0-10 μM). (C) Representative western blot of global H3K27me3 levels following 6 or 24 h of GSK-J4 treatment (5 μM). (D) Metaplots of spike-in normalized H3K27me3 CUT&RUN signal in DMSO-treated HEK293T cells and HEK293T cells treated with 5 μM GSK-J4 for 6 h or 24 h. Inhibition of KDM6A/B results in a global increase in promoter H3K27me3 levels. (E) Principal component analysis (PCA) plot of spike-in normalized TimeLapse-seq read counts, transformed by the rlog function in DESeq2. DMSO- and 24 h GSK-J4-treated samples clustered separately.

Article Snippet: Unless otherwise indicated in the figure captions, experiments targeting KDM6A/B were performed by treating HEK293T cells with 5 μM GSK-J4 (MCE, HY-15648B) for either 6 or 24 hrs.

Techniques: Binding Assay, Western Blot, Inhibition, Transformation Assay