Gene regulation by nuclear hormone receptors in vivo
Author: Mansén, Anethe
Date: 2003-12-05
Location: CMB auditorium, Berzelius väg 21, Karolinska Institutet
Time: 9.00
Department: Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
Abstract
The nuclear hormone receptor superfamily contains 49 different members, including those for thyroid hormone, TRα and TRβ. These receptors are ligand-modulated transcription factors that activate or repress gene activity. The TRα gene encodes two main splice variants, TRα1 and TRα2. Furthermore, the orphan receptor Rev-erbAα gene is located juxtaposed to and partially overlapping the TRα gene, which has led to the hypothesis that transcription of one of them could interfere with expression of the other. In this thesis work I have studied the consequences for target gene expression and normal physiology of targeting the TR or the Rev-erbAα genes in the mouse genome.
The targeting of the Rev-erbAα gene by insertion of a β-gal cassette resulted in the appearance of novel transcripts for both Rev-erbAα and TR. Despite this, the TRα1 to TRα2 expression ratio was unaltered. On the other hand, the Rev-erbAα gene was found to negatively regulate its own expression, as shown by the β-gal activity in homozygote tissue being eight times higher than it is in heterozygotes and wt. Deletion of Rev-erbAα was also associated with a delayed cerebellar maturation. The results show that Rev-erbAα expression is important for an appropriate balance between transcriptional activation and repression during postnatal cerebellar development.
TRα2-deficient mice were generated using a different targeting approach: introduction of a strong polyadenylation site that blocks transcription into the TRα2 coding exon. This generated a shorter TRα1 transcript that was, however, expressed at increased levels. Both TRα2+/- and -/- mice had a complex phenotype with both hypothyroid and hyperthyroid features, attributable to the elevated expression of TRα1. This suggested that the ratio of TRα1 to TRα2 is important for maintaining an adequate physiological response to TH.
TR ablation was shown to affect the regulation of several T3 responsive genes in the heart. Myosin heavy chain β expression, which normally is suppressed by T3-bound TR, was highly overexpressed in TRα1 -/- mice. However, it was suppressed by T3 in both TRα1 -/- and TRâ -/- mice, indicating that both isoforms can mediate repression. In contrast, the expression of MyHCα was not significantly affected by the deletion of either TRα or TRβ. Thus we conclude that the TR isoforms have distinct specificities in gene regulation of the cardiac MyHC genes.
In our study of how T3 affects cardiac function, we found that the KCNE1 ion channel gene is negatively regulated by T3-activated TRα1. Transgenic mice overexpressing KCNE1 via a heart-specific promoter had very similar cardiac abnormalities to those of the TRα1 -/- mice. This supports the hypothesis that dysregulation of the KCNE1 gene can cause the cardiac dysfunction of TRα1 mice. Electrophysiological experiments confirmed that elevated KCNE1 levels perturb ion current activities, suggesting that the stoichiometry of the KCNE1 β-subunit relative to other ion channel α-subunits is of vital importance for heart function.
The targeting of the Rev-erbAα gene by insertion of a β-gal cassette resulted in the appearance of novel transcripts for both Rev-erbAα and TR. Despite this, the TRα1 to TRα2 expression ratio was unaltered. On the other hand, the Rev-erbAα gene was found to negatively regulate its own expression, as shown by the β-gal activity in homozygote tissue being eight times higher than it is in heterozygotes and wt. Deletion of Rev-erbAα was also associated with a delayed cerebellar maturation. The results show that Rev-erbAα expression is important for an appropriate balance between transcriptional activation and repression during postnatal cerebellar development.
TRα2-deficient mice were generated using a different targeting approach: introduction of a strong polyadenylation site that blocks transcription into the TRα2 coding exon. This generated a shorter TRα1 transcript that was, however, expressed at increased levels. Both TRα2+/- and -/- mice had a complex phenotype with both hypothyroid and hyperthyroid features, attributable to the elevated expression of TRα1. This suggested that the ratio of TRα1 to TRα2 is important for maintaining an adequate physiological response to TH.
TR ablation was shown to affect the regulation of several T3 responsive genes in the heart. Myosin heavy chain β expression, which normally is suppressed by T3-bound TR, was highly overexpressed in TRα1 -/- mice. However, it was suppressed by T3 in both TRα1 -/- and TRâ -/- mice, indicating that both isoforms can mediate repression. In contrast, the expression of MyHCα was not significantly affected by the deletion of either TRα or TRβ. Thus we conclude that the TR isoforms have distinct specificities in gene regulation of the cardiac MyHC genes.
In our study of how T3 affects cardiac function, we found that the KCNE1 ion channel gene is negatively regulated by T3-activated TRα1. Transgenic mice overexpressing KCNE1 via a heart-specific promoter had very similar cardiac abnormalities to those of the TRα1 -/- mice. This supports the hypothesis that dysregulation of the KCNE1 gene can cause the cardiac dysfunction of TRα1 mice. Electrophysiological experiments confirmed that elevated KCNE1 levels perturb ion current activities, suggesting that the stoichiometry of the KCNE1 β-subunit relative to other ion channel α-subunits is of vital importance for heart function.
List of papers:
I. Chomez P, Neveu I, Mansen A, Kiesler E, Larsson L, Vennstrom B, Arenas E (2000). Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor. Development. 127(7): 1489-98.
Pubmed
II. Salto C, Kindblom JM, Johansson C, Wang Z, Gullberg H, Nordstrom K, Mansen A, Ohlsson C, Thoren P, Forrest D, Vennstrom B (2001). Ablation of TRalpha2 and a concomitant overexpression of alpha1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol Endocrinol. 15(12): 2115-28.
Pubmed
III. Mansen A, Yu F, Forrest D, Larsson L, Vennstrom B (2001). TRs have common and isoform-specific functions in regulation of the cardiac myosin heavy chain genes. Mol Endocrinol. 15(12): 2106-14.
Pubmed
IV. Mansen A, Tiselius C, Sand P, Rydqvist B, Vennström B (2003). Expression of the KCNE1 ion channel subunit is negatively regulated by thyroid hormone receptor alpha1. [Submitted]
I. Chomez P, Neveu I, Mansen A, Kiesler E, Larsson L, Vennstrom B, Arenas E (2000). Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor. Development. 127(7): 1489-98.
Pubmed
II. Salto C, Kindblom JM, Johansson C, Wang Z, Gullberg H, Nordstrom K, Mansen A, Ohlsson C, Thoren P, Forrest D, Vennstrom B (2001). Ablation of TRalpha2 and a concomitant overexpression of alpha1 yields a mixed hypo- and hyperthyroid phenotype in mice. Mol Endocrinol. 15(12): 2115-28.
Pubmed
III. Mansen A, Yu F, Forrest D, Larsson L, Vennstrom B (2001). TRs have common and isoform-specific functions in regulation of the cardiac myosin heavy chain genes. Mol Endocrinol. 15(12): 2106-14.
Pubmed
IV. Mansen A, Tiselius C, Sand P, Rydqvist B, Vennström B (2003). Expression of the KCNE1 ion channel subunit is negatively regulated by thyroid hormone receptor alpha1. [Submitted]
Issue date: 2003-11-14
Publication year: 2003
ISBN: 91-7349-678-2
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