Fig. 5: ANLN regulates gene transcriptional activity and Pol II chromatin binding.

A Volcano plots depicting gene expression changes in control and ANLN knockdown KYSE150 cells. The false discovery rates (FDRs) are extracted from DESeq2 by adjusting P-values using empirical Bayes approach. B, C Metascape database was used for enrichment analysis of differential genes in RNA-Seq data. Based on the selected terms, accumulative hypergeometric P-values and enrichment factors were calculated and used for filtering. Remaining significant terms were hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. The 0.3 kappa score was applied as the threshold to cast the tree into term clusters. D, E Proportion of 2-fold differentially expressed genes with or without ANLN bound within 3 kb of TSS (D), and the average signal intensity of ANLN occupancy (E). n = 168 (Down); n = 75 (Up). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. F, G Metaplots and heatmaps showing the occupancy of H3K27ac and total Pol II measured by CUT&Tag in NC and ANLN short-term depletion (24 h) cells. H Representative track examples of down-regulated genes (TXNRD1, CCND1) and housekeeping genes (GAPDH) showing the occupancy of H3K27ac and Pol II after short-term depletion of ANLN. I FPKM of representative genes in RNA-Seq data. n = 3 for each group. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. J Western blot showing the whole cell lysate, chromatin pellets, and chromatin supernatant in ANLN short-term depletion cells. Pellets and supernatant were obtained by eluting chromatin components with 400 mM NaCl followed by centrifugation. K pGLC or pGLB plasmids were transfected into Dox-induced ANLN knockdown KYSE150 cells or control cells. Cells were treated with Dox 24 h after transfection, and luciferase activity was measured 48 h after transfection. Renilla luciferase activity served as an internal control. n = 4 for each group. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. L–N Metaplots comparing the average H3K27ac signal in NC and ANLN short-term depletion cells. Flow chart for identifying signals in the super-enhancer regions of KYSE150 cells (L). CUT&Tag signal is shown for typical enhancer (M) and super-enhancer regions (N). CUT&Tag analysis representative of two independent experiments, and other data are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.