AbstractBackgroundThe transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysisResultsExhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes.ConclusionOur study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte. ; We acknowledge the support of Grants BFU-2010-16025 from the Dirección General de Proyectos de Investigación; RD06/0020/0060 from FIS, Instituto de Salud Carlos III, and S2011/BMD-2328 TIRONET project from the Comunidad de Madrid (Spain). S. Ruiz-Llorente holds a postdoctoral fellowship of the Instituto de Salud Carlos III (Contrato Postdoctoral de Perfeccionamiento; CD05-0055). A. Sastre-Perona holds a predoctoral fellowship from the Formación Personal Universitario (FPU) program. C. Montero-Conde holds a postdoctoral fellowship of the Spanish Ministry of Science and Innovation (MICINN; BMED2008-0659).