Identification of thyroid hormone receptor binding sites in developing mouse cerebellum

Thyroid hormones play an essential role in early vertebrate development as well as other key processes. One of its modes of action is to bind to the thyroid hormone receptor (TR) which, in turn, binds to thyroid response elements (TREs) in promoter regions of target genes. The sequence motif for TREs remains largely undefined as does the precise chromosomal location of the TR binding sites. A chromatin immunoprecipitation on microarray (ChIP-chip) experiment was conducted using mouse cerebellum post natal day (PND) 4 and PND15 for the thyroid hormone receptor (TR) beta 1 to map its binding sites on over 5000 gene promoter regions. We have performed a detailed computational analysis of these data.

By analysing a recent spike-in study, the optimal normalization and peak identification approaches were determined for our dataset. Application of these techniques led to the identification of 211 ChIP-chip peaks enriched for TR binding in cerebellum samples. ChIP-PCR validation of 25 peaks led to the identification of 16 true positive TREs. Following a detailed literature review to identify all known mouse TREs, a position weight matrix (PWM) was created representing the classic TRE sequence motif. Various classes of promoter regions were investigated for the presence of this PWM, including permuted sequences, randomly selected promoter sequences, and genes known to be regulated by TH. We found that while the occurrence of the TRE motif is strongly correlated with gene regulation by TH for some genes, other TH-regulated genes do not exhibit an increased density of TRE half-site motifs. Furthermore, we demonstrate that an increase in the rate of occurrence of the half-site motifs does not always indicate the specific location of the TRE within the promoter region. To account for the fact that TR often operates as a dimer, we introduce a novel dual-threshold PWM scanning approach for identifying TREs with a true positive rate of 0.73 and a false positive rate of 0.2. Application of this approach to ChIP-chip peak regions revealed the presence of 85 putative TREs suitable for further in vitro validation.

This study further elucidates TRβ gene regulation in mouse cerebellum, with 211 promoter regions identified to bind to TR. While we have identified 85 putative TREs within these regions, future work will study other mechanisms of action that may mediate the remaining observed TR-binding activity.