Isoform-specific transcriptional activity of overlapping target genes that respond to thyroid hormone receptors alpha1 and beta1 - PubMed (original) (raw)

Isoform-specific transcriptional activity of overlapping target genes that respond to thyroid hormone receptors alpha1 and beta1

Ivan H Chan et al. Mol Endocrinol. 2009 Nov.

Abstract

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of physiology and development. TRs are expressed in vertebrates as a series of distinct isoforms that exert distinct biological roles. We wished to determine whether the two most widely expressed isoforms, TR alpha 1 and TR beta 1, exert their different biological effects by regulating different sets of target genes. Using stably transformed HepG2 cells and a microarray analysis, we were able to demonstrate that TR alpha 1 and TR beta 1 regulate a largely overlapping repertoire of target genes in response to T(3) hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T(3)-mediated regulation by TR alpha 1 than by TR beta 1, to near equal regulation by both isoforms. We also identified TR alpha 1 and TR beta 1 target genes that were regulated by these receptors in a hormone-independent fashion. We suggest that it is this gene-specific, isoform-specific amplitude of transcriptional regulation that is the likely basis for the appearance and maintenance of TR alpha 1 and TR beta 1 over evolutionary time. In essence, TR alpha 1 and TR beta 1 adjust the magnitude of the transcriptional response at different target genes to different levels; by altering the ratio of these isoforms in different tissues or at different developmental times, the intensity of T(3) response can be individually tailored to different physiological and developmental requirements.

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Figures

Figure 1

Figure 1

Stable ectopic expression of TRα1 and TRβ1 in HepG2 cells results in enhanced T3-dependent gene activation. A, TRα1 and TRβ1 mRNA levels in HepG2 cells stably transformed with TRα1, TRβ1, or empty [no receptor (NR)] expression plasmids. Total RNA from each pool of stable HepG2 cells was isolated, and TR mRNA levels were determined by quantitative RT-PCR. Relative copy number was calculated as a ratio of TR to GAPDH expression used as an internal normalization control (mean +

sem

; n = 3). B, Enhanced T3-dependent reporter activation in TRα1 and TRβ1 transformants. HepG2 cells stably integrated with TRα1, TRβ1, or empty plasmid control (NR) were transiently transfected with a DR4-TK-luciferase reporter and a pCH110 construct as an internal control. The cells were treated with or without T3 24 h later as indicated and harvested 48 h after transfection, and the luciferase activity was determined relative to β-galactosidase activity (mean +

sem

; n = 3).

Figure 2

Figure 2

TRα1 and TRβ1 regulate overlapping sets of T3-responsive genes. A, Schematic of pairwise comparisons employed: HepG2 cells stably transformed by TRα1, by TRβ1, or by the empty plasmid control were treated with vehicle only or with T3 for 6 h. RNA was isolated, and the genes in each transformant were up- or down-regulated by T3 were identified in the following pairwise comparisons: TRα1 transformants minus vs. plus T3, TRβ1 transformants minus vs. plus T3, and empty plasmid (NR) transformants minus vs. plus T3. B, Venn diagram of genes regulated by T3 in TRα1 transformants, by T3 in TRβ1 transformants, or by T3 in the empty plasmid control (NR), identified at a Benjamini-Hochberg (BH) adjusted P value of <0.05. The number of T3-regulated genes shared, or not, among the three different transformants are enumerated and are shown schematically as overlapping circles. C, The same genes identified as T3-up-regulated in panel B are presented again, but at a BH-adjusted P value of <0.1. NR, No receptor.

Figure 3

Figure 3

T3 response of representative target genes. A–J, Microarray intensity signal values of mRNAs, minus and plus T3, for genes flagged as T3-regulated in the TRα1, TRβ1, or empty plasmid transformants (adjusted P < 0.05; mean +

sd

; n = 3). Asterisk indicates that the difference between minus and plus T3 was significant at a P value ≤0.05. NR, No receptor.

Figure 4

Figure 4

Validation of microarray data by RT-PCR analysis and heat map of genes regulated by T3 in TRα1 or TRβ1 transformants. A–D, The mRNAs from TRα1, TRβ1, or empty plasmid (NR) transformants, minus or plus 6 h of T3 treatment, were subjected to RT-PCR using the gene-specific primers described in Materials and Methods. The resulting PCR products were stained with SYBR Green, and the gel band intensity values were quantified using an 8900 gel documentation system (Alpha Innotech, San Leandro, CA). The level of expression of each gene product was defined as 1 for the empty-vector control in the absence of T3 (mean +

sem

; n = 3). E, Heat map clustering of genes up-regulated by T3 in the HepG2 TRα1, TRβ1, or empty plasmid transformants (P <0.05). Dark blue indicates low expression, dark red indicates high expression, with intermediate values represented by lighter shades. *, The difference between minus and plus T3 was significant at a P value ≤0.05; #, the difference between minus and plus T3 was significant at a P value ≤0.1. NR, No receptor.

Figure 5

Figure 5

A subset of genes are down- or up-regulated by TRα1 or TRβ1 irrespective of T3 status. A, Schematic of pairwise comparisons employed: HepG2 cells stably transformed by TRα1, by TRβ1, or by the empty plasmid control were treated with vehicle only or with T3 for 6 h. RNA was isolated, and the genes down- or up-regulated by the presence of the ectopic TRα1 or TRβ1 were identified in the following pairwise comparisons: TRα1 transformants vs. empty plasmid (NR) transformants in the absence of T3, TRβ1 transformants vs. empty plasmid (NR) transformants in the absence of T3, TRα1 transformants vs. empty plasmid (NR) transformants in the presence of T3, TRβ1 transformants vs. empty plasmid (NR) transformants in the presence of T3. B, Venn diagram of genes down-regulated in TRα1 transformants compared with the empty plasmid (NR) control, or down-regulated in TRβ1 transformants compared with the empty plasmid (NR) control regardless of T3 concentration. All were flagged using a Benjamini-Hochberg (BH) adjusted P value of <0.05. The number of down-regulated genes shared, or not, between the TRα1 and TRβ1 transformants is enumerated and is shown schematically as overlapping circles. C, Venn diagram of genes up-regulated in TRα1 transformants compared with the empty plasmid (NR) control, or up-regulated in TRβ1 transformants compared with the empty plasmid (NR) control regardless of T3 concentration. All were flagged using a Benjamini-Hochberg (BH) adjusted P value <0.05. The number of up-regulated genes shared, or unshared, between the TRα1 and TRβ1 transformants is enumerated and is shown schematically as overlapping circles. NR, No receptor.

Figure 6

Figure 6

Validation of microarray data by RT-PCR analysis: genes down- or up-regulated by TRα1 or TRβ1 regardless of T3 status. A–F, The mRNAs from TRα1, TRβ1, or empty plasmid (NR) transformants, minus or plus 6 h of T3 treatment, were subjected to RT-PCR using the gene-specific primers described in Materials and Methods. The resulting PCR products were stained with SYBR Green, and the gel band intensity values were quantified using an 8900 gel documentation system (Alpha Innotech). The level of expression of each gene product was defined as 1 for the empty-vector control in the absence of T3 (mean +

sem

; n = 3). *, The difference between vector only and plus the TR isoform was significant at a P value ≤ 0.05; #, the difference was significant at a P value ≤0.1. NR, No receptor.

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