Fig 1.
Microtubule damage triggers DGE.
(A-D) DGE in RPE1 hTert cells treated with microtubule poisons for 6 (a and b) or 24 h (c and d). Data are represented as average Log2FCs from three independent biological replicates, relative to basal expression in DMSO-treated control cells. Plotted are expression profiles of genes that have p-value < 0.05 and >50 mRNA reads per million, in 1 nM CA4- and 3 nM PTX-treated cells (a and c) and 100 nM CA4- and 300 nM PTX-treated cells (b and d). In red are depicted genes with FDR < 0.02 for low drug doses and < 0.001 for high drug doses, of which names are printed for top 20 genes. Plot quadrants are labeled from I–IV. (E and F) GSEA based on expression profiles of genes from quadrants II and IV, from cells treated with high drug doses for 6 (b) or 24 h (d). Plotted are −Log10 BH-adjusted p-values of the top five enriched GO terms in the category Biological Process, with BH-adjusted p-value < 0.01. ACTB, β-actin; ACTN, actinin; ANKRD, Ankyrin Repeat Domain; BH, Benjamini–Hochberg; CA4, combretastatin A-4; CCDC, Coiled-coil domain containing; COX, Cytochrome C Oxidase; CTGF, Connective Tissue Growth Factor; CYR, Cytochrome Reductase; CYTB, Cytochrome B; DEG, differentially expressed gene; DGE, differential gene expression; EGR, Early Growth Response; ERRF, Endoplasmatic Reticulum-Related Factor; FDR, false discovery rate; GO, gene ontology; GSEA, gene set enrichment analysis; hTert, human telomerase reverse transcriptase; KHDRBS, KH RNA Binding Domain Containing, Signal Transduction Associated; LBH, Limb Bud And Heart Development; Log2FC, Log2 Fold Change; MALAT, Metastasis-Associated Lung Adenocarcinoma Transcript; MAP, microtubule-associated protein; MEG, Maternally Expressed Gene; ND, NADH Dehydrogenase; NEAT, Nuclear Paraspeckle Assembly Transcript; NKTR, Natural Killer Cell Triggering Receptor; OGT, O-Linked N-Acetylglucosamine Transferase; PTX, paclitaxel; RPE1, retinal pigment epithelial 1; SCG, Secretogranin; SLC, Solute Carrier Family; SRF, Serum Response Factor; SRM, Spermidine Synthase; THBS, Thrombospondin; TMX, Thioredoxin Related Transmembrane Protein; TM4SF, Transmembrane 4 L Six Family Member; TNFRSF, Tumor Necrosis Factor Receptor Superfamily Member; TTC, Tetratricopeptide Repeat Domain; TUBA, α-tubulin; TUBB, β-tubulin; XIST, X Inactive Specific Transcript.
Fig 2.
Microtubule damage triggers differential expression of all TUBA and TUBB isoforms.
(A, left panel) Expression profiles of all detected TUBA and TUBB isoforms in our DGE data set. Dendrogram on the left represents Pearson distance between expression profiles. Each column of the heatmap represents DGE in one treatment, labeled on the x-axis above and below the heatmap, relative to DMSO control. Each row represents a gene, labeled on the y-axis. In bold are differentially expressed genes that have p-value < 0.05. Color key is depicted in upper left corner. Data are represented as Log2FC relative to DMSO control. (A, right panel) Average Log2 mRNA CPMs in DMSO-treated control samples are presented as a bar chart. (B and C) Relative abundance of unspliced TUBA1A and TUBB pre-mRNA in control (DMSO) and cells treated with microtubule poisons (x-axis) for 6 (B) or 24 h (C). (D and E) Relative abundance of spliced TUBA1A and TUBB mRNA in control (DMSO) and cells treated with microtubule poisons (x-axis) for 6 (D) or 24 h (E). All the expression profiles are normalized to a reference gene (GAPDH or RPL19) and to DMSO control. Error bars in all panels represent standard deviation from three independent biological replicates. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001 in paired Student t test compared to DMSO control. CA4, combretastatin A-4; CPM, count for each gene per million detected reads; DGE, differential gene expression; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Log2FC, Log2 Fold Change; PTX, paclitaxel; RPL19, ribosomal protein L19; TUBA, α-tubulin; TUBB, β-tubulin; TUBD, δ-tubulin; TUBE, ε-tubulin; TUBG, γ-tubulin.
Fig 3.
Cells coordinate expression of TUBA and TUBB isoforms.
(A) Scheme of the bioinformatic approach. (B and C) Pearson expression correlation coefficients for a subset of abundantly expressed TUBA and TUBB isoforms across 417 human (B) and 122 mouse (C) publicly available Affymetrix chip data sets. In dark red are genes that show strong expression correlation (Pearson correlation coefficient = 1), and in gray are genes that show no expression correlation (Pearson correlation coefficient = 0). Color keys are represented to the left of each heatmap. High expression correlation for a subset of tubulin genes is marked as cluster CEM1. Tubulin genes that do not correlate in expression are marked as Null module. (D) Frequency diagram of groups of perturbations among the top 100 data sets with the highest tubulin gene-expression correlation from the human platform. CEM, Co-Expressed Modules; GEO, Gene Expression Omnibus; TUBA, α-tubulin; TUBB, β-tubulin; TUBD, δ-tubulin; TUBE, ε-tubulin; TUBG, γ-tubulin.
Table 1.
The top 100 data sets with highest tubulin-gene–expression Pearson correlation from the human platform.
Annotated are studies rank-ordered by decreasing tubulin-gene–expression Pearson correlation, with their associated GEO identifiers. Extracted from the CLIC report is differential tubulin gene expression. Unless NA, down-regulated (down) or up-regulated (up) tubulin mRNA levels are annotated. Based on published literature, perturbations in the listed studies are annotated into 11 groups, and their effect on microtubule cytoskeleton is marked as known (yes) or unknown (no).
Fig 4.
PI3K activity increases tubulin gene expression through autoregulation.
(A) DGE in parental and cells expressing constitutively active PI3K H1047R mutant cells, treated with DMSO or indicated PI3K inhibitors for 4 h. (B and C) Relative expression of TUBA1A and TUBB unspliced pre-mRNA (B) and spliced TUBA1A and TUBB mRNA (C) in parental and PI3K mutant cell lines H1047R and E545K, treated with DMSO control or 1 μM GDC-0941 for 4 h. All the relative gene-expression data are normalized to housekeeping gene GAPDH or RPL19 and to DMSO-treated parental cells. (D) Tubulin partitioning to unpolymerized (soluble, S) and polymerized (P) normalized to loading control (GAPDH for S; HISH3 for P) and to DMSO-treated parental cells in parental and cells expressing constitutively active PI3K mutants H1047R and E545K, treated with DMSO or 1 μM GDC-0945 for 4 h. (E) DGE in A2058 cells treated with DMSO control or the indicated concentrations of PI3K inhibitors for 6 h (GEO series GSE66343 [28]). (F–G) RT-qPCR in A2058 cells treated with 1 μM GDC-0941 for indicated periods of time (x-axes). (H) Tubulin partitioning to unpolymerized (S) and polymerized (P) normalized to loading control (GAPDH for S; HISH3 for P) and to DMSO-treated cells in control and cells treated for 6 h with indicated 1 μM PI3K inhibitors. (I) Representative immunofluorescence images of control and cells treated with 1 μM GDC-0941 for 6 h and stained with anti-EB1 antibody (cyan), anti-β-actin, and Hoechst (red). (J) Relative number of detected EB1 comets per cell area in DMSO control and A2058 cells treated with 1 μM GDC-0941 for 6 h, normalized to DMSO-treated cells (100–200 cells). (K) A proposed model describes the mechanism through which PI3K signaling regulates tubulin gene expression through modification of tubulin autoregulation. Bar plots in all panels represent average values, and error bars standard deviations from three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 in paired Student t test, in all panels. Black and white asterisk represent statistical significance relative to DMSO-treated parental cell line, green asterisks represent statistical significance relative to DMSO-treated control of the same cell line. a.u., arbitrary unit; DGE, differential gene expression; EB1, end-binding protein 1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; HISH3, histone H3; pAKT, phospho-AKT; PI3K, phosphatidylinositol-4,5-biphosphate 3-kinase; RPL19, ribosomal protein L19; RT-qPCR, reverse-transcription quantitative PCR; TUBA, α-tubulin; TUBB, β-tubulin; TUBD, δ-tubulin; TUBE, ε-tubulin; TUBG, γ-tubulin; WT, wild type.