Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum

Abstract

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

Figure 1. Assessing specificity of antibody and ChIP protocol.

A. Characterization of TRβ antibody with the GH3 cell line. A single 52 kDa band was detected with Western blot. B. Characterization of the enrichment of MBP-TRE in all cerebellum samples. Enrichment of fragments from the promoter region of β-actin and MBP was examined with PCR using IP and TI DNA amplified with WGA kit. One representative sample in the bottom shows the enrichment in non-amplified DNA. The right panel shows the enrichment ratio of MBP to β-actin calculated using quantified band intensities.

Figure 2. Distribution of genomic locations of binding sites of 91 genes.

The mid point of each probe was used to calculate the distance to the closest gene.

Figure 3. Examples of TRβ binding activities identified with ChIP-on-chip.

The plots show enrichment ratios for all probes within a genomic region (IP versus TI DNA). Chromosomal positions are from NCBI build 34 of the mouse genome. The start and direction of transcription are noted by arrows. Black bars on the X axis indicate the fragments whose enrichment was confirmed with PCR.

Figure 4. Confirmation of enriched genes identified using ChIP-on-chip with 13 randomly selected genes by PCR in independently prepared amplified IgG-IP, TRβ-IP and TI DNAs pool of 2 samples.

Figure 5. The expression of novel thyroid responsive genes in hypothyroid or hyperthyroid mouse models.

A. MMI/perchlorate induced hypothyroid, hyperthyroid or hypothyroid/replacement animal models. B. PTU induced hypothyroid animal models. RT-PCR was performed with RNA extracted from cerebellum on PND15 (n = 5). * Significantly different from control (p<0.05).

Figure 6. The transcriptional activity of the MAG promoter examined with the luciferase reporter assay.

A. The location of the 3 truncated fragments used to build the reporter constructs. The PCR-confirmed ChIP enriched fragment is indicated by the black bar. The TSS and direction is indicated with an arrow. B and C. Transcriptional activity of GH reporter construct or MAG promoter reporter constructs induced by TH. The reporter constructs and empty vector were co-transfected into GH3 cells with pRL-TK (as a transfection efficiency control). T3 (10⁻⁸ M) was added after 24 hrs. Firefly luciferase expression was normalized to renilla luciferase from the pRL-TK plasmid. Values are mean±S.E. (n = 3). *indicates p<0.05.