Barhl1 is directly regulated by thyroid hormone in the developing cerebellum of mice.
Thyroid hormones (THs) are essential for the brain development. Despite considerable effort, few genes directly regulated by THs have been identified. In this study, we investigate the effects of THs on the regulation of Barhl1, a transcription factor that regulates sensorineural development. Using DNA microarray combined with chromatin immunoprecipitation (ChIP-chip), we identified a TRβ binding site in the promoter of Barhl1. The binding was further confirmed by ChIP-PCR. The site is located approximately 755 bp upstream of the transcription start site. Reporter vectors containing the binding site or mutated fragments were transfected into GH3 cells. T3 treatment decreased the transcriptional activity of the wild fragment but not the mutant. Two 28 bp oligonucleotides containing sequences that resemble known TH response elements (TREs) were derived from this binding site and DNA-protein interaction was performed using electrophoretic mobility shift assays (EMSA). Binding analysis in a nuclear extract containing TRβ revealed that one of these fragments bound TRβ. This complex was shifted with the addition of anti-TRβ antibody. We investigated Barhl1 expression in animal models and TH-treated cultured cells. Both long term treatment with 6-propyl-2-thiouracil and short-term treatment with 0.05% methimazole/1% sodium perchlorate (both treatments render mice hypothyroid) resulted in up-regulation of Barhl1. TH supplementation of hypothyroid mice caused a decrease in the expression of Barhl1 compared to control animals. Similarly, the expression of Barhl1 in cultured GH3 decreased with the addition of T3. Given the important role of Barhl1 in brain development, we propose that perturbations of TH-mediated transcriptional control of Barhl1 may play a role in the impaired neurodevelopment induced by hypothyroidism.
Hazard Identification Division, Environmental Health Science and Research Bureau, Health Canada, 50 Columbine Driveway, Ottawa, Ontario, Canada K1A 0K9. hongyan_dong@hc-sc.gc.ca
Hongyan
H
Dong
Carole L
CL
Yauk
Michael G
MG
Wade
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D005260:Female; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D055495:Neurogenesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D020218:Response Elements / Q000187:drug effects; D013963:Thyroid Hormones / Q000378:metabolism* / Q000494:pharmacology
C485866:Barhl1 protein, mouse; D009419:Nerve Tissue Proteins; D012097:Repressor Proteins; D013963:Thyroid Hormones