Genes & Development



Supplemental Material and MethodsRelated to Supplemental Fig. S1ELISASerum total T4 and T3 hormones levels were measured by an ELISA kit purchased from Alpha Diagnostic, San Antonio, TX. Serum TSH was measured by MILLIPLEX MAP rat Thyroid Magnetic Bead Panel (EMD Millipore) by the PENN Radioimmunoassay and Biomarkers core.Western BlotLiver samples were lysed in a TissueLyser (Qiagen) in radioimmunoprecipitation assay buffer (RIPA buffer) supplemented with complete EDTA-free protease inhibitor (Roche) and 1 mM PMSF. Samples were resolved by Tris-glycine SDS-PAGE (Biorad), transferred to nitrocellulose membrane (Biorad), and blotted with the anti-HA antibody 3F10 High Affinity (Roche, 1:1000).Immunofluorescence Livers were dissected from mice and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Slides were rehydrated and subjected to antigen retrieval in sodium citrate (pH 6.0). H2O2 (30%) was used for quenching endogenous peroxidases. Incubation with primary antibody anti-HA C29F4 (Cell signaling technologies, 1:50) was done overnight at 4°C in a humid chamber, followed by secondary antibody incubation for 2 hours at room temperature.Quantitative polymerase chain reaction (qPCR)RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed with Power SYBR Green PCR Master Mix and a QuantStudio 6 Flex instrument (Applied Biosystems), and analysis was performed by the standard curve method. All qPCR data were normalized to Rsp9 expression. Error bar represents the standard error of the mean (SEM). The primers sequences are: Thrb: Fw 5’-TCCAGTCCTTGGTGTGAATG and Bw 5’-GAAATGCTGTTTGGCTTGCT, Dio1: Fw 5’-GATTGTTGGTGATAGGTGTTTCC and Bw 5’-GCCTGCGATTTGGTTTAGTT, Thrsp: Fw 5’-GAGAACGACGCTGCTGAAA and Bw 5’-GGTGGGTAAGGATGTGATGG, Rsp9: Fw 5’-CATCCTTCATTGTTCGCCT and Bw 5’-TGGCATTCTTCCTCTTCACTC.Related to Supplemental Fig. S5CistromeDB and motif analysis of coregulatorsTo compare NCoR1, HDAC3, and CBP datasets against CistromeDB, we first called differential peaks between PTU and T3 conditions using HOMER’s getDifferentialPeaks command two ways and then merging the results. Then these peaks were submitted to CistromeDB Toolkit web service, obtaining GIGGLE scores for 200 most similar ChIP-seq experiments in the database.Related to Supplemental Fig. S8Hi-C data analysisProcessed chromatin contact matrix (at ZT10) and main TAD list were downloaded from GEO under accession GSE104129. Contact strength cutoff was set at 10. TRBS regions were intersected with 5Kbp segments of the contact matrix, and the statistics of contacts were collected. 1000 random control regions were sampled uniformly from the whole genome.For TAD clustering analysis, regions of interest (genes or TRBS) were counted in each TAD. The resulting counts statistics were compared to probabilities under binomial distribution, assuming random distribution of these regions across TADs. The statistical test used is Monte-Carlo simulation (1 million samples) of the exact null model, using the XNomial R package.For TAD-TAD contacts analysis, we collect all contacts between different TADs. We then calculate the relative frequency of contacts within a certain group of TADs (TADs containing CBP/NCoR up- and downregulated TRBS), and frequency of contacts outside of this group. ................
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