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Molecular basis of thyroid hormone regulation of myelin basic protein gene expression in rodent brain
A Farsetti1, T Mitsuhashi, B Desvergne
1Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Regulation of myelin basic protein (MBP) gene expression by thyroid hormone has been investigated in rodent brain. Quantitation of the 4 major alternatively spliced transcripts by RNase protection assay showed that the individual mRNAs, corresponding to MBP isoforms 21.5, 18.5, 17, and 14 kDa, were decreased from 2- to 17-fold at all ages studied (4-60 days) in hypothyroid animals when compared to euthyroid, but the timing of onset of expression was not altered. MBP mRNA was also reduced in young adult rats thyroidectomized at the age of 5-6 weeks and was restored to normal by thyroxine administration. Nuclear run-off assays showed that the rate of MBP gene transcription is dependent on thyroid state. Co-transfection of MBP (-256/+1)-chloramphenicol acetyltransferase chimeric gene with a plasmid expressing thyroid hormone receptor alpha, and in the presence of 3,5,3'-triiodothyronine, into NIH3T3 or NG108-15, increased chloramphenicol acetyltransferase expression 4-fold. Using a footprinting technique and Spodoptera frugiperda 9 (Sf9) nuclear extract infected with baculovirus expressing TR alpha, we have identified a single DNA-binding site (-186/-163) for the receptor. A part of this region contains the AGGACA sequence found in thyroid hormone-responsive elements of other 3,5,3'-triiodothyronine-regulated genes. Our finding of a specific hormone-receptor interaction with the MBP promoter region is the first direct demonstration of a thyroid hormone-responsive element in a brain-specific gene.
Insights
Thyroid hormone regulates myelin basic protein (MBP) gene expression in rodent brains. This study identifies a specific DNA-binding site for the thyroid hormone receptor on the MBP gene promoter, demonstrating direct hormonal regulation of this brain-specific gene.
Area of Science:
- Neuroscience
- Molecular Biology
- Endocrinology
Background:
- Thyroid hormone is crucial for brain development and function.
- Myelin basic protein (MBP) is a key component of myelin, essential for nerve impulse conduction.
Purpose of the Study:
- To investigate the role of thyroid hormone in regulating myelin basic protein (MBP) gene expression in the rodent brain.
- To identify the molecular mechanisms underlying thyroid hormone's effect on MBP gene transcription.
Main Methods:
- Quantification of alternatively spliced MBP mRNA transcripts using RNase protection assays.
- Analysis of MBP gene transcription rates using nuclear run-off assays.
- Reporter gene assays (chloramphenicol acetyltransferase) and DNA footprinting to identify hormone-receptor interactions on the MBP promoter.
Main Results:
- Hypothyroidism significantly decreased MBP mRNA levels (2- to 17-fold) across various ages in rodents.
- Thyroidectomy reduced MBP mRNA, which was restored by thyroxine administration.
- Nuclear run-off assays confirmed thyroid hormone's regulation of MBP gene transcription rate.
- A specific thyroid hormone receptor alpha binding site (-186/-163) was identified on the MBP promoter, containing a known hormone-responsive element sequence.
Conclusions:
- Thyroid hormone directly regulates MBP gene expression in the rodent brain.
- A specific DNA-binding site for the thyroid hormone receptor on the MBP promoter has been identified.
- This finding provides the first direct evidence of a thyroid hormone-responsive element in a brain-specific gene.