Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: RNA synthesis is downregulated but gene transcription persists in nucleoplasm despite Pol-II depletion. POLR2A-AID2 Degron line was treated with auxin, DMSO, or Actinomycin D treated (5 µg/mL) for 6 hours and imaged following labeling of nascent RNA with 1 mM 5-ethynyl-uridine for 1 hour. ( A ) Representative image of each condition captured using widefield fluorescent microscopy. Scale bar = 10 µm. ( B ) Cell total corrected fluorescence of widefield fluorescent images for each condition. Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001) (POLR2A: N = 142; 6hrActD: N=20; 1hrActD: N=28; DMSO: N= 204). ( C-D ) Representative images from SMLM of EU-labeled RNA for each condition. ( E-G ) Quantification of SMLM distributions for each condition generated using DBscan: fluorophore blink density, cluster density, and cluster size (POLR2A: N = 18; DMSO: N= 28). Significance was calculated by unpaired t test with Welch’s correction applied. P-values are listed. ( H-I ) MA plot for POLR2A degraded cells and ActD treated cells showing log2 expression as a function of log fold change using a LFC cutoff of (abs > 1) and signficance level of Padj < 0.05. X axis = Log2 Mean Expression (averaged over all samples). ( J ) Scatterplot of log fold chance values for each gene in one condition compared to log fold change values for the same gene in another condition. Heatmap color scale corresponds to gene density. Marginal distribution shows DEGs with same cutoffs as MA plots. For B, E– G : Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116-POLR2A-AID2 cells were treated with 1 μM 5-phenyl-indole-3-acetic acid (5- Ph-IAA; MedChemExpress, HY-134653) to degrade endogenous POLR2A.

Techniques: Labeling, Microscopy, Fluorescence, Generated, Expressing

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Genome connectivity and in situ packing domains are perturbed by Pol-II loss. POLR2A-AID2 degron line was treated with auxin for 6 hours, DMSO for 6 hours, or Actinomycin D treated (5 µg/mL) for 1 hour and Hi-C was generated for each condition. ( A-C ) Scatterplot of log10 loop strength against log10 size for each loop. Loops called using HICCUPS. Heatmap shows loop density (POLR2A Loops: N =56,420; DMSO Loops: N=18,123; ActD Loops: N=39,484). ( D ) Top: difference plot showing change relative to WT. Scale bar: normalized mean loss or gain of contact strength. Bottom: Pileup plots of loop insulation strength for each. Scale bar: normalized mean contact strength. Insulation plots for loop strength were generated in GENOVA . ( E ) TAD insulation plots showing no change to TADs between conditions in POLR2A depleted cells. TADs called using Arrowhead and plotted with GENOVA ( F ) Contact map highlighting loop loss upon POLR2A depletion. Red arrows mark loops that have decreased focal enrichment. ( G ) Contact map from reanalyzed Micro-C data showing distal loop gain and proximal loop loss following POLR2A depletion. Red arrows mark loops that have increased focal enrichment. Yellow arrows mark loops that have decreased focal enrichment. ( H) Compartment pile-up plot showing no change to compartments (both TAD and compartment pileup plots were generated using GENOVA. See methods for more details). ( I) Contact scaling for individual genes binned and ranked by E/I score (E/I = Exon Length/(Gene Length – Exon Length) ). Accompanying schematic shows how E/I scales with domain size. Low E/I = large domains, average E/I = small domains, and high E/I = chained aggregates that have are less likely to form domains on their own. (J) Schematic demonstrating how the ratio of exons to introns (E/I ratio) within genes organizes domain geometry. Genes with a low E/I ratio are more likely to form domains, wherein heterochromatic introns form sticky cores that exclude euchromatic exons in a transcriptionally poised superficial ideal zone for coherent gene transcription. For I : Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116-POLR2A-AID2 cells were treated with 1 μM 5-phenyl-indole-3-acetic acid (5- Ph-IAA; MedChemExpress, HY-134653) to degrade endogenous POLR2A.

Techniques: In Situ, Hi-C, Generated, Insulation

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Loss of Pol-II triggers packing domain core degradation and heterochromatin swelling. SMLM data was generated for POLR2A-AID2 line treated with auxin for 6 hours or DMSO for 6 hours ( A ) Representative images of SMLM for labelled H3K9me3 (blue) and labelled active POLR2A (red). ( B ) Association analysis of POLR2A with H3K9me3 clusters showing that POLR2A associates with dense packing domain cores. ( C-D ) Representative images of H3K9me3-labeled SMLM. Scale bar = 2 µm. ( E-F ) Quantification of H3K9me3-conjugated SMLM distributions for each condition: DBscan cluster density and fluorophore blink density, respectively. Data was compiled from two independent biological replicates (DMSO: N = 50; POLR2A: N= 53). ( G ) H3K9me3- conjugated SMLM clusters organized by cluster size and density. In each size category, cluster density increases for auxin treated cells. ( H ) Schematic showing H3K9me3 ChIP-seq peak signal as a function of domain CVC and intron length. Longer introns have more H3K9me3 and contribute to larger domains with a higher CVC. ( I ) Publicly available H3K9me3 ChIP-seq data for a POLR2A-AID2 line treated with auxin for 6 hours or DMSO for 6 hours was analyzed by gene length ( J ) and then by intron length. For G, I-J, Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001).

Article Snippet: HCT116-POLR2A-AID2 cells were treated with 1 μM 5-phenyl-indole-3-acetic acid (5- Ph-IAA; MedChemExpress, HY-134653) to degrade endogenous POLR2A.

Techniques: Generated, Labeling, ChIP-sequencing

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Loss of Pol-II triggers large packing domain degradation and proliferation of small domains. POLR2A-AID2 line was treated with auxin for 6 hours or DMSO for 6 hours and assessed using ChromSTEM. ( A-C ) PWS following treatment: average nuclear packing scaling, diffusion, and fractional moving mass of packing domain nuclear average, respectively. Data was compiled from three independent biological replicates. (DMSO: N = 539; POLR2A: N= 570; ActD: N=475). ( D ) ChromSTEM tomograms showing intact packing domains in untreated cells or degraded packing domain in cells with POLR2A loss. XY axes are in nm. ( E ) Analysis of packing domains by size and packing efficiency to analyze domain properties. Nascent domains (low efficiency, small size), large and small mature domains (high packing efficiency), and decaying domains (low efficiency, large size) represented by color. Lines represent median packing efficiency and median radius in control cells. Each dot represents a single domain. Bar chart in right corner shows number gained or lost in each category (POLR2A: N = 61; DMSO: N=86). ( F ) Publicly available ATAC-seq data for a POLR2A- AID2 line treated with auxin for 6 hours or DMSO for 6 hours was analyzed by Exon/Intron ratio (E/I) for each gene. Exon/Intron ratio corresponds to the surface area to volume ratio (SA/V) of domains ( G ) Schematic showing Log2 E/I as a function of domain SA/V. Low E/I values have large domains and vice versa. For A-C, F , Significance was calculated by unpaired t test with Welch’s correction applied (**** < 0.0001). For E , confidence intervals on bar plot were calculated using a Wilson Score Test.

Article Snippet: HCT116-POLR2A-AID2 cells were treated with 1 μM 5-phenyl-indole-3-acetic acid (5- Ph-IAA; MedChemExpress, HY-134653) to degrade endogenous POLR2A.

Techniques: Diffusion-based Assay, Control

Journal: bioRxiv

Article Title: Gene transcription and chromatin packing domains form a self- organizing system

doi: 10.64898/2026.03.15.711889

Figure Lengend Snippet: Loss of Pol-II upregulates intronic regions within gene bodies and triggers 5’ readthrough at gene transcription end sites. Analysis of intronic and intergenic expression in RNA-seq generated from POLR2A-AID2 degron line treated with auxin or DMSO for 8 hours or ActD (5 µg/mL) for 6 hours. ( A ) RIP-seq signal on gene bodies for POLR2A immunoprecipitated transcripts in HCT116 cells showing that Pol-II is enriched on the TSS and TES of genes. Region: ± 1kb. ( B ) Intron and exon signal from transcripts immunoprecipitated with an EZH2 (control) or POLR2A antibody in WT HCT116 cells was averaged across 20 bins for all gene bodies. Coverage was normalized, log2 transformed, and plotted. Pol-II RIP in shows gain of signal on both introns and exons. ( C ) Boxplot showing distribution of nascent (EU-seq) differentially expressed genes in POLR2A degraded cells (abs > 0.58) ( D-G ) Representative genomic loci where intron upregulation or 5’ transcriptional readthrough was observed. Red arrows denote 5’ readthrough regions. Orange arrows denote intron upregulation at loci. Coverage was RPGC normalized and Log2 transformed for ease of visualization. ( H ) For nascent transcripts, intron and exon signal was averaged across 20 bins for all gene bodies. Coverage was normalized, log2 transformed, and plotted for each condition by transcript length. ActD shows loss of nascent signal in exons and introns. Pol-II depleted cells show little loss of nascent signal overall, while intron signal decreases with transcript length.

Article Snippet: HCT116-POLR2A-AID2 cells were treated with 1 μM 5-phenyl-indole-3-acetic acid (5- Ph-IAA; MedChemExpress, HY-134653) to degrade endogenous POLR2A.

Techniques: Expressing, RNA Sequencing, Generated, Immunoprecipitation, Control, Transformation Assay

Journal: Cells

Article Title: Phosphoproteome Remodeling upon CDK1 Inhibition Restricts HSV-1 IE Gene Transcription and Replication

doi: 10.3390/cells15050407

Figure Lengend Snippet: Backbone protein–protein interaction network under CDK1 inhibition during HSV-1 infection. Differentially phosphorylated proteins from the HSV-1/CDK1i vs. HSV-1/DMSO comparison were mapped to STRING (Homo sapiens; highest confidence, combined score ≥ 0.9) and imported into Cytoscape; node centralities were computed with cytoHubba. The backbone was defined as the union of nodes in the top decile for MCC, Degree, or Betweenness. CDK1 is shown at the center (red). Proteins annotated as transcription-related are arranged in the right ring (yellow); the remaining backbone proteins are grouped on the left into functional clusters based on curated Biological Processes. Nodes highlighted in green are known in vitro and in vivo CDK1 targets (motif-based). Edges depict STRING functional/physical interactions; line intensity reflects the STRING combined score; POLR2A is indicated with a black arrow. Programming language R (version 4.4.2) and RStudio integrated development environment (version 2024.12.0+467) were used to generate this image with select labels added using Microsoft PowerPoint 2016.

Article Snippet: POLR2A hypophosphorylation was monitored with primary antibodies specific to total POLR2A (Invitrogen, Carlsbad, CA, USA, cat. #MA1-10882; 1:1500 dilution) and phosphorylation-specific antibodies: phospho-Ser2-POLR2A (Novus Biologicals, Centennial, CO, USA, NBP2-59215; 1:1500 dilution) and phospho-Ser5-POLR2A (Abcam, Waltham, MA, USA, ab5408; 1500 dilution).

Techniques: Inhibition, Infection, Comparison, Functional Assay, In Vitro, In Vivo

Journal: Cells

Article Title: Phosphoproteome Remodeling upon CDK1 Inhibition Restricts HSV-1 IE Gene Transcription and Replication

doi: 10.3390/cells15050407

Figure Lengend Snippet: CDK1 inhibition decreases RNAPII CTD phosphorylation during early HSV-1 infection. ( a ) Schematic of RNAPII (POLR2A) CTD heptads 38–48 highlighting residues that were hypophosphorylated from our phosphoproteomic dataset (red boxes; approximate decreases shown in red, sites correspond to S2/T4/S4/S5 of the YSPTSPS consensus). ( b ) Immunoblot of POLR2A, Ser2-phosphorylated POLR2A (p-S2), and Ser5-phosphorylated POLR2A (p-S5) in infected cells treated with DMSO or CDK1i. β-actin served as a loading control; “−/+” indicates the absence/presence of CDK1i or HSV-1. Treated and untreated lanes shown are infected replicates. ( c ) Densitometry analyses of p-S2-POLR2A and p-S5-POLR2A; bands of the phosphorylated POLR2A were normalized to total POLR2A. For all panels, HFFs were pre-treated with CDK1i for 1 h, infected with HSV-1 (KOS, MOI = 2), and harvested at 3 hpi; M, PageRuler™ Plus Prestained Protein Ladder. Microsoft Excel 2016 and Microsoft PowerPoint 2016 were used to generate this figure.

Article Snippet: POLR2A hypophosphorylation was monitored with primary antibodies specific to total POLR2A (Invitrogen, Carlsbad, CA, USA, cat. #MA1-10882; 1:1500 dilution) and phosphorylation-specific antibodies: phospho-Ser2-POLR2A (Novus Biologicals, Centennial, CO, USA, NBP2-59215; 1:1500 dilution) and phospho-Ser5-POLR2A (Abcam, Waltham, MA, USA, ab5408; 1500 dilution).

Techniques: Inhibition, Phospho-proteomics, Infection, Western Blot, Control