Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A. Schematic representation of the 5-EU metabolic labeling strategy. Cells were treated with 1 mM 5-ethynyl uridine (5-EU) for 10 min to label nascent RNA, followed by biotinylation (biotin-azide) via click chemistry and streptavidin-based affinity purification to enrich proteins. B Western blot validation of the 5-EU enrichment strategy. C4-2 cells were labeled with 5-EU for 10 minutes and then enriched according to the procedure in this study. The final eluent was analyzed by Western blotting using antibodies against RNA Pol II and GAPDH. Non-labeled cells served as negative control. C Venn diagram showing the overlap and specificity of proteins identified by 5-EU enrichment versus RNA Pol II immunoprecipitation (IP). D Gene Ontology (GO) and E KEGG enrichment analysis of proteins unique to the 5-EU and RNA Pol II IP datasets using enrichr .

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques: Labeling, Affinity Purification, Western Blot, Biomarker Discovery, Negative Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: Volcano plots showing differentially expressed genes (DEGs) in C4-2 cells treated with THZ531 (200 nM) for 4 h and 24 h. Volcano plots show significantly differentially expressed genes (DEGs) in C4-2 cells under different inhibitor treatments and at different treatment durations. Conditions: 500 nM YKL-5-124 (4 h), 20 nM NVP-2 (4 h), 200 nM THZ531 (4 h), 100 nM THZ531 (24 h), 200 nM THZ531 (24 h).

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques:

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Metagene plots of ATAC-seq signals centered on transcription start sites (TSS) in C4-2 cells treated with THZ531 for 4 h (acute phase) and 24 h (late phase), showing loss of chromatin accessibility. B Transcription factor (TF) motif analysis in regions losing accessibility at 4 h, showing loss of general TFs like SP1 and KLF. C Quantification of differentially accessible regions (DARs). Bar plot shows the number of significantly closed regions (FDR < 0.05) upon CDK12 inhibition (24h) compared to controls. D Integration of RNA-seq and ATAC-seq data. The violin plot shows that CDK12 inhibition suppresses the expression of long genes in DARs and promotes the expression of short genes in DARs. Significance was assessed using Student’s t -test. Gene length grading is consistent with . E Four-quadrant plot correlating changes in chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) with CDK12 inhibition. Left: CDK12 inhibition primarily affects the downregulation of significantly close genes. A four-quadrant plot showing changes in chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) after CDK12 inhibition (200 nM THZ531, 24h) reveals that significantly closed-down (6454) genes constitute the largest proportion. Right: ATAC-seq signal trajectory visualization shows that in closed genes under THZ531 treatment, the ATAC signal at the PRKDC and RAD51 sites in long genes related to DNA repair remained unchanged after 4h treatment but were more closed after 24h treatment. F Motif analysis of TF binding sites in chromosome 24h after THZ531 (CDK12/13) treatment shows a significant enrichment of CTCF motif loss.

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques: Inhibition, RNA Sequencing, Expressing, Gene Expression, Binding Assay

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Volcano plot of RNA Pol II-interacting proteins in C4-2 cells treated with 150 nM THZ531 (CDK12/13i), 20 nM NVP-2 (CDK9i) and 500 nM YKL-5-124 (CDK7i) for 4 h. The results show that 127, 43 and 72 proteins are significantly enriched under CDK12i, CDK9i and CDK7i, respectively, and 62, 18 and 12 proteins are significantly decreased (p < 0.05 and log2FC ± 1). B Upset plots compare the distribution of proteins that are significantly lost and gained after inhibition of CDK7, CDK9, or CDK12 under the 5-EU metabolic labeling method. C The dotplot shows the GO molecular function enrichment analysis of proteins that were significantly increased after inhibition of CDK7, CDK9, and CDK12. CDK12 inhibition specifically upregulated and enriched the “Damaged DNA Binding” and “Histone Acetylation” pathways, while CDK7i and CDK9i do not. D The protein-protein interaction (PPI) network (STRING database) of the adaptive complex formed around RNA Pol II, which exhibits significant increased interaction with RNA Pol II upon CDK12 inhibition, is centered on DDB1, BRD4, PAF1, and ELOA.

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques: Inhibition, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Prostate cancer cells depend on major transcription-associated cyclin-dependent kinases (CDK7, CDK9 and CDK12), whereas normal prostate cells show relative resistance to inhibitors targeting these kinases. Each cell line was cultured for 24 hours before treatment and then treated for 4 days in 50 nM YKL-5-124, 20 nM NVP-2 and 150 nM THZ531, respectively, and their cell viability was assessed using CellTiterGlo. The data is from three biological replicates, each having three technical replicates. B The results show that in the C4-2 cell line, PAF1 knockdown produced significant combined decrease in viability only with CDK12 inhibition. DDB1 knockdown produced significant combined decrease in viability with CDK12 and CDK9 inhibition, but not with CDK7 inhibition. BRD4 knockdown produced significant combined decrease in viability with CDK12, CDK9, and CDK7 inhibition. In the 22RV1 cell line, DDB1 and BRD4 knockdown produced combined decrease in viability with CDK12, CDK9, and CDK7 inhibition, but PAF1 knockdown produced significant combined lethality only with CDK12 inhibition. Cell viability after 4 days of DDB1, BRD4, and PAF1 knockdown with CDK7, CDK9, and CDK12 inhibitor treatments for the last three days. Data were obtained from three biological replicates (each containing three technical replicates), and the standard error of the mean was calculated. Student’s t -test was used to assess significance. C The knockdown of DDB1, BRD4, and PAF1 was confirmed by western blotting in castration-resistant prostate cancer cell lines C4-2 and 22RV1 (n=2).

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques: Cell Culture, Knockdown, Produced, Inhibition, Western Blot

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: Cell viability was assessed using CellTiter-Glo 4 days after ELOA and PLK3 knockdown, followed by inhibition with CDK7, CDK9, and CDK12 inhibitors for the last 3 days (the doses are indicated in the figure). Data were obtained from 3 biological replicates (each biological replicate contained 3 technical replicates), and the standard error of the mean was calculated. Significance was assessed using Student’s t -test. A Viability assay shows that knockdown of ELOA or PLK3 induce combinatorial anti-proliferative effects with CDK12, CDK9, and CDK7 inhibition, respectively, in the 22RV1 cell line. B and C Viability assays show that knockdown of ELOA or PLK3 do not induce combined lethality with CDK12, CDK9, and CDK7 inhibition in the C4-2 and LNCaP cell lines. D Viability assay shows that knockdown of ELOA or PLK3 do not trigger combined lethality with CDK12, CDK9, and CDK7 inhibition in the normal prostate cell line PNT1.

Article Snippet: C4-2, 22RV1 and LNCaP cell lines were obtained from the American Tissue Culture Collection, and the PNT1 cell line was acquired from Sigma.

Techniques: Knockdown, Inhibition, Viability Assay