Open Access Research article

Genome-wide analysis of Pax8 binding provides new insights into thyroid functions

Sergio Ruiz-Llorente12, Enrique Carrillo Santa de Pau134, Ana Sastre-Perona1, Cristina Montero-Conde12, Gonzalo Gómez-López3, James A Fagin2, Alfonso Valencia3, David G Pisano3 and Pilar Santisteban1*

Author Affiliations

1 Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas (CSIC) y Universidad Autónoma de Madrid (UAM), C/Arturo Duperier 4, Madrid, 28029, Spain

2 Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA

3 Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

4 Department of Molecular Biology, Faculty of Science, Nijmegen Centre of Molecular Life Sciences, Radboud University, Nijmegen, HB, 6500, The Netherlands

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BMC Genomics 2012, 13:147  doi:10.1186/1471-2164-13-147

Published: 24 April 2012

Additional files

Additional file 1:

(A). Pax8 chromatin immunoprecipitation validation performed to confirm enrichment of target DNA fragments by means of real-time PCR. Sequences belonging to the Nis upstream enhancer element (NUE) [7] and Tpo promoter sequences [9], previously described as Pax8 binding sites in rat thyroid cells, were used as positive controls. (B). ChIP-Seq results with regard to the Nis locus were visualized in the UCSC genome browser. Significant immunoprecipitated peak corresponding to MACS program included the Nis upstream enhancer (NUE), previously described to be regulated by Pax8 (underlined red letters).

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Additional file 2:

ChIP-Seq MACS data: Excel file containing genomic coordinates of 13,151 rat genomic regions significantly immunoprecipitated according to the MACS ChIP program.

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Additional file 3:

ChIP seq peaks used for MEME/TOMTOM consensus motif analysis: Genomic coordinates of the 500 most significant ChIP peaks used to verify Pax8-dependent immunoprecipitation and to delineate the consensus Pax8 DNA binding site.

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Additional file 4:

DNA binding motifs overrepresented in Pax8 IP peaks. Genomatix matrices significantly associated to Pax8 MACS peaks. Overrepresentation (genome) column represents overrepresentation values of these matrices in our IP peaks compared to their presence along the rat genome, and Z-Score (genome) column indicates association value of Pax8-immunoprecipitated DNA for each considered DNA matrix.

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Additional file 5::

Expression data of wt and scrambled conditions vs. siPax8 conditions and association with ChIP-Seq data.Common probes (+/− 1kb) Excel sheet includes expression data for the 78 probes common to both expression comparisons (wt and siScramble conditions vs. siPax8; p<0.005) and belonging to genes showing a significant IP peak within 1kb of a TSS. Additional Excel sheets include significant probes for each array comparison.

Format: XLSX Size: 8.7MB Download file

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Additional file 6:

FatiScan gene set enrichment analysis. Excel file containing Gene Ontology (GO) terms commonly overrepresented in both expression array comparisons (datasheet “Common GO terms") and their adjusted p-values. This file also contains datasheets showing GO terms statistically significant for each individual comparison (WT or SCR sign GO biol. process).

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Additional file 7:

FatiScan gene set enrichment analysis for scrambled and wild type conditions vs. siPax8 conditions. FatiScan image showing enriched GO terms in siScramble vs. siRNAPax8 and wt vs. siRNAPax8 comparisons, respectively.

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Additional file 8:

FatiScan gene set enrichment analysis for scrambled and wild type conditions vs. siPax8 conditions. FatiScan image showing enriched GO terms in siScramble vs. siRNAPax8 and wt vs. siRNAPax8 comparisons, respectively.

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Additional file 9:

Significant biological processes among underexpressed probes. FatiGO images showing overrepresented biological processes among common downregulated (n=633) probes for both expression array comparisons.

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Additional file 10:

Significant biological processes among overexpressed probes. FatiGO images showing overrepresented biological processes among common upregulated (n=565) probes for both expression array comparisons.

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Additional file 11:

Overrepresented KEGG pathways among under- and overexpressed probes. KEGG pathways enriched among common downregulated (n=633) and upregulated (n=565) probes for both expression array comparisons.

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Additional file 12:

UCSC genome browser images showing significant Pax8 IP peaks for closely positioned loci which were detected to be significantly deregulated in expression arrays (p<0.005).

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Additional file 13:

Oligonucleotides used for immunoprecipitation validation prior to performing high throughput sequencing (including positive and negative Pax8 immunoprecipitation controls), or for experimental validation of ChIP-Seq.

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Additional file 14:

Schematic representation of experimental design followed for whole genome rat expression arrays. Both comparisons (PCCl3-siPax8 vs. PCCl3-wt and PCCl3-siPax8 vs. PCCl3-siScramble) included four different biological replicates that were cross-labelled with either Cy3 or Cy5.

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Additional file 15:

Immunoblot demonstrating Pax8 downregulation in siPax8 conditions (siPax8) versus control conditions, including the no transfection condition (wt) and siScramble PCCl3-transfected cells (siScramble). Time points include 24 and 48 hours.

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Additional file 16:

Oligonucleotides used for experimental validation of expression arrays.

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