Figure 1.
A. Equal expression of exogenously expressed TRs. Upper panel, western blot of extracts of HepG2 parental cells (1), HepG2 cells infected with control lentivirus (2) or cells infected with lentivirus expressing TRα (3) or TRβ (4) and blotted with anti-flag antibody. Inset beneath shows the same extracts blotted with a β-actin antibody as a loading control. Lower panel; western blot of HeLa-TR extracts after +/− doxycycline withdrawal to induce TRs and blotted with anti-myc. B. Results of T3 binding assays performed on extracts of HeLa-TR cells after 24 hrs doxycycline withdrawal; figures in panels represent deduced affinities of expressed TRs for T3. C. Results of luciferase assays performed upon HeLa-TR cells transfected with standard TRE-driven reporters, DR-4 Luc and IP-6 (F2)-Luc after doxyclycline withdrawal to induce TR expression. D. Western blot of HepG2-TR extracts at various times after initial T3 treatment.
Figure 2.
A–D. Numbers of genes that meet cut-offs for fold induction and statistical significance in parental HepG2, HepG2-TRα or HepG2-TRβ at each time point, A, 3 hr, B, 6 hr, C, 24 hr, D, all three time points combined. T3 induced genes are represented in upper panels (red) and T3 repressed genes in lower panels (blue), note the difference in scale which means that many more genes are positively regulated than negatively regulated. E–H. Plots of fold induction/repression by T3 in the presence of TRβ (y-axis) versus TRα (x-axis). E, 3 hr blue, F, 6 hr green, G, 24 hr red, H, all three time points.
Figure 3.
Heatmap to Illustrate Patterns of T3 Response in HepG2.
A. Representation of changes in all T3-dependent genes that meet cutoffs for fold induction/statistical significance in HepG2 parental cells and HepG2-TRα or HepG2-TRβ cells at each time point. Red, upregulated, green, downregulated, black, no change. Genes were clustered according to similarities in response patterns as described in Methods. B. As for Fig. 3A, with a section of the heatmap expanded to reveal one of the clusters of late emerging TRβ-preferential T3 responses.
Figure 4.
Verification of TRβ preference of late responding genes.
qPCR analysis of T3 induction of two genes (A, G6Pc and B, GSTA1) identified as preferential late TRβ responders. Note that TRα-responses are weak and that TRβ preference persists across all timepoints.
Figure 5.
T3 Induced Genes are Direct TR Targets.
A. Bar graph representing numbers of T3 induced (upper panels) and repressed (lower panels) genes at 3 hrs in HepG2-TRα and HepG2-TRβ cells that persist with CHX pre-treatment (upper panel, red, lower panel, blue). B. Heat map representing gene expression changes at 3 hrs timepoint in HepG2-TRα and HepG2-TRβ cells with T3, CHX and T3 + CHX. Note that most target genes retain their T3 responses with CHX. Examples of genes with unusual responses are marked by lower case letters: a = stronger T3 responses with TRα, b = amplification of weak T3 responses in the presence of TRβ with CHX, c = selective CHX-dependent gene induction in the presence of TRα, d = selective CHX-dependent gene induction in the presence of TRβ.
Figure 6.
Verification of different T3 response patterns in HepG2 cells.
Results of qPCR analysis of representative gene expression changes at various times after T3 induction in HepG2-TRα and HepG2-TRβ cells. A, PCK1, similar with both TRs, B, SLCA16A, similar with both TRs at most times, C, HIF2A, TRβ preference at both early and late times, D, Myh6, TRα preference at early and late times.
Figure 7.
A. Numbers of genes that meet cutoffs for fold T3 activation (upper panel, blue) or repression (lower panel, red) in HeLa-TRα and Hela-TRβ cells at 24 hrs treatment, as in Fig. 2. B. Plots of fold induction/repression by T3 in the presence of TRβ (y-axis) versus TRα (x-axis) in HeLa cells. C–D. Representative qPCR analysis showing examples of different gene regulation patterns with the two TRs. C, pck1, D, thrsp.
Figure 8.
Partial overlap of T3 regulated genes in HepG2 and HeLa.
Venn diagrams of numbers of T3 induced and repressed genes identified in each cell type with TRα and TRβ and overlaps.
Figure 9.
Gene expression changes with unliganded TRs.
A. Numbers of genes that meet fold cutoffs for activation/repression and statistical significance in response to unliganded TR expression in HepG2 cells, TRα and TRβ expressing cells were compared to parental. B. HeLa cells, TRα and TRβ expressing cells after doxycycline withdrawal to induce TRs versus doxycyclin treated cells. Similar results were obtained in comparisons with parental HeLa cells (not shown). C. Plots of fold induction/repression by TRβ (y-axis) versus TRα (x-axis) in HepG2 cells. D. Plots of fold induction/repression by TRβ (y-axis) versus TRα (x-axis) in HeLa cells.
Figure 10.
Hypothetical patterns of TR regulation.
Genes with statistically significant responses to T3 or unliganded TRs were assigned into categories according to net repression (R), induction (I) or no change (O) represented in the schematic heat map. Numbers of genes in each category and overlaps between genes that respond to TRα or TRβ in this manner are shown in Table 1.
Table 1.
Patterns of TR regulation.
Figure 11.
Unusual ligand-independent TR gene-regulation patterns.
qPCR verification of genes that display hormone-independent repression by both TRs in HepG2. A, mst1, B, hel308.
Figure 12.
Verification of TR subtype preferences in gene regulation pattern.
A, Myh6 B, furin. Both genes display the same pattern of response to unliganded TRs and T3, despite preferential T3 induction of Myh6 with TRα. C, ALPI, display exclusively ligand-dependent induction with TRα and ligand-dependent induction with TRβ coupled to a strong ligand-independent component. D, HIF2A, displays the opposite profile to ALPI in Fig. 12C.
Table 2.
T3 concentration dependence of gene induction.