Journal: bioRxiv

Article Title: Epromoters bind key stress-related transcription factors to regulate clusters of stress response genes

doi: 10.1101/2024.11.26.625372

Figure Lengend Snippet: (A) Schematic representation of the pipeline steps to identify Epromoter-regulated gene clusters. (B) Schematic overview of the two clustering methods used in the pipeline. (Left) Clustering of stress-induced genes based on the proximity of their TSS within a 100 kb distance. (Right) Clustering of stress-induced genes based on their localization within the same TAD. (C) Schematic representation of the “Vihervaara” dataset to induce the HS response. (D) (left) Bubble plot showing the number of all the clusters identified by their number of promoters (x-axis) compared to their number of promoters recruiting HSF1 (y-axis) determined by the pipeline. (right) Bubble plot showing the number of all the clusters identified by their number of promoters (x-axis) compared to their number of promoters recruiting HSF2 (y-axis) determined by the pipeline. (E) Venn diagram displaying the overlap of Epromoter-regulated clusters identified after clustering either by the distance between the TSS or their localization within the same TAD in the HS response. (F) Example of the PGGHG Epromoter-regulated cluster identified by the pipeline with the two clustering methods in the HS stress response. The genomic tracks show the PRO-seq signal (red) and HSF1 and HSF2 ChIP-seq signal (blue) before or after HS from the “Vihervaara” dataset. The topological associating domains (TAD) from K562 is displayed on the top. The fold-change of induction is indicated below the name and orientation of the genes (Epromoter: green, co-induced genes: yellow). (G) Luciferase assays to quantify the enhancer activity of predicted Epromoter (on the right) and induced promoters clustered with Epromoters (on the left) before (blue) and after (red) HS in the K562 cells. The results were normalized on the pGL4-SV40 promoter plasmid. P values were calculated by a paired one-sided Student’s t-test. (insert) Luciferase assay-induced enhancer activity represented by the fold change before/after HS for the predicted Epromoter and the induced promoters. P values were calculated by a two-sided Wilcoxon’s test, *** P < 0.001, ** P < 0.01, * P < 0.1. (H) (top panel) Representation of the classification of the HS-induced genes in 4 categories. (bottom panel) Percent stacked barplot of the numbers of motifs (HSE) per promoter in each category. The number of motifs is divided into 0 motif (blue), 1 motif (cyan), 2 to 3 motifs (yellow), and more than 3 motifs (red).

Article Snippet: Cell line K562 (CCL-243), a chronic myelogenous leukemia cell line, was obtained from the ATCC (American Type Culture Collection) and maintained in RPMI 1640 media (Thermo Fischer Scientific) supplemented with 10% FBS (Thermo Fischer Scientific) at 37°C and 5% CO2.

Techniques: ChIP-sequencing, Luciferase, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Epromoters bind key stress-related transcription factors to regulate clusters of stress response genes

doi: 10.1101/2024.11.26.625372

Figure Lengend Snippet: (A) The NUCB1 Epromoter-regulated cluster. (top) Hi-C triangular matrix (resolution 5kb) of the locus from K562 cells. The genomic tracks show the PRO-seq signal (in red) and HSF1 and HSF2 ChIP-seq signal (in blue) before or after HS from the “Vihervaara” dataset. The fold-change of induction is indicated below the name and orientation of the genes (Epromoter: green, co-induced genes: yellow). (B) Luciferase assays to quantify the promoter activity of the induced DHDH promoter with or without the NUCB1 Epromoter acting as an enhancer before and after HS in K562 cells. P values were calculated by a paired one-sided Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.1. (C) Schematic of the CRISPR-Cas9 genome deletion and re-insertion of the promoter region of the NUCB1 Epromoter in K562 cells. (d) qPCR analysis of TULP2 , NUCB1 , and DHDH expression in wild type and 4 ΔNUCB1 mutants in K562 cells before and after HS. Values represent the relative expression of the samples normalized to the housekeeping gene GAPDH and compared to the unstressed wild-type cells. P values were calculated by a two-sided Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.1. (E) qPCR analysis of the DHDH expression in 2 ΔNUCB1 mutants, 2 heterozygote ΔNUCB1-inserted mutants (green, blue), and 2 homozygote ΔNUCB1-inserted mutants in K562 cells before and after HS. Values represent the relative expression of the samples normalized to the housekeeping gene GAPDH , then compared before and after HS. P values were calculated by a two-sided Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.1. (F) qPCR analysis of the promoter activity in 2 ΔNUCB1 mutants, 2 heterozygote ΔNUCB1-inserted mutants, and 2 homozygote ΔNUCB1-inserted mutants in K562 cells before and after HS. Values represent the relative expression of the samples normalized to the housekeeping gene GAPDH and compared to the mean of the ΔNUCB1 clones. P values were calculated by a two-sided Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.1.

Article Snippet: Cell line K562 (CCL-243), a chronic myelogenous leukemia cell line, was obtained from the ATCC (American Type Culture Collection) and maintained in RPMI 1640 media (Thermo Fischer Scientific) supplemented with 10% FBS (Thermo Fischer Scientific) at 37°C and 5% CO2.

Techniques: Hi-C, ChIP-sequencing, Luciferase, Activity Assay, CRISPR, Expressing, Clone Assay