Despite the known relevance of the transcription factor Pax8 for adult thyrocyte physiology, few data have been published concerning Pax8 target genes other than key thyroid-related genes (Tg
Tpo, and Nis). The transcriptional output of Pax8 during thyroid development is unknown but essential, given that thyroid follicular precursors are not formed in Pax8 null mouse embryos, which ultimately impairs the formation of follicle structures and thyroid hormone biosynthesis .
With regard to its link to tumour development, Pax8 expression decreases or is lost in follicular thyroid carcinomas as well as in oncogene-transformed thyroid cells . Moreover, several well-known tumour suppressors, including TP53 and WT1, have been defined as Pax8 targets, and cytoplasmic Pax8 staining has been positively associated with tumour size, metastasis, local invasion, recurrence, or persistence in the thyroid . Taking into account all these premises, and in order to better understand the role of Pax8 in the maintenance of thyroid function, we decided to explore the transcriptional profile of Pax8-silenced thyroid PCCl3 cells, and to integrate these signals with genome wide cis-regulatory studies. Thus, our experimental design combined putative and novel Pax8 binding sites with analysis of actual target gene expression regulation, a strategy successfully used for identifying direct targets for other transcription factors [35, 36].
Our unbiased mapping of Pax8 binding sites along the rat genome has identified a large number of DNA sequences that are occupied in living thyrocytes. Moreover, this is the first study addressing in vivo genome-wide mapping of Pax8-DNA binding sites, and the Pax8 consensus binding motif here defined encompasses motifs described by previous reports focused either on single gene regulation [7, 12] or on Paired-box DNA motif characterization [13, 14]. The ChIPSeq approach also led to significant immunoprecipitation of genomic sequences containing CpG islands, as well as CpG dinucleotides. Extensive literature has linked the location of CpG islands and GC-enriched regions to transcriptionally permissive chromatin [37, 38], which could lend support to a relevant role of Pax8 in the transcriptional output of the thyrocyte. About half of all CpG islands self-evidently contain TSSs, while the other half (known as “orphan” CpG islands) are either within or between characterized transcription units and have unknown significance [11, 38]. Despite a lack of association to annotated promoters, “orphan” CpG islands have been associated to transcriptional initiation and dynamic expression during development . In agreement with this, we found significant Pax8 binding to orphan CpG islands in intronic regions and a preferential binding to such islands 10–100 kb upstream or downstream of a transcription start site. In fact, genomic studies indicate that almost half of the human coding genes have alternative promoters  and that transcription factor binding sites (TFBSs) in classically defined promoter regions may represent a minority of genomic binding sites . Moreover, this latter report clearly demonstrated an association between TFBSs and the expression of non-coding RNAs, which could be modulating the expression of the gene encoded by the opposite strand. Less directly, a subset of intergenic H3K4me3 peaks, many of which are likely to correspond to orphan CpG islands, were found to represent TSSs for long non-coding RNAs . Our findings suggest that Pax8 binds orphan CpG islands that could represent alternative promoters of nearby annotated genes  or ncRNAs that regulate gene expression.
Otherwise, Pax8-dependent ChIP-Seq data demonstrated an enrichment of genomic regions with overrepresentation of general transcriptional regulatory elements (Human MTE and Drosophila MTE, Inr-DPE and BRE). MTE constitutes a core promoter element (~20-30 nt downstream of the TSS) associated with RNA polymerase II-mediated transcription [44, 45]. Furthermore, human orphan CpG islands have been associated with RNA polymerase II binding sites . On the other hand, Inr-DPE and BRE elements represent functional binding sites for TFIIB and TFIID (transcription initiation factor IIB and IID, respectively), which are main components of the basal transcription machinery . Interestingly, Jin et al recently described synergistic MTE-Inr-BRE transcriptional modules in more than 9,000 orthologous mouse and human genes . Whereas functional experiments should be performed to demonstrate an interaction of Pax8 with these general core elements, our data underscore the importance of synergistic interactions between core promoter elements and tissue-specific TFs to ultimately modulate gene expression.
Potential Pax8 partners in transcriptional regulation
2Apart from the classical view of TFs interacting with promoter regions, TFs could activate gene expression by interacting with common lineage-specific TFs and/or binding to distal regions (enhancers). Synergistic effects of Pax8 and AP1 proteins have been shown to occur in the regulation of Nis transcription through interaction along the NUE element , and AP1 and PAX proteins also interact to cooperate in the modulation of transcription of other genes . Accordingly, we observed an overrepresentation of binding motifs related to NRF-1 (Nuclear respiratory factor-1), and several AP1 members (c-FOS, BATF3, and c-JUN) were differentially expressed in Pax8-deprived thyroid cells. However, no significant findings were obtained for other transcription factors described to act synergistically with Pax8, such as Nkx2-1 and TAZ/WWTR1 proteins , indicating that this cooperative transcriptional role could be restricted to specific loci rather than representing a global transcription phenomenon in thyroid cells.
Functional studies described in the present paper confirmed physical in vivo interactions between Pax8 and CTCF or Sp1 in thyrocytes. These novel partners were further demonstrated to modulate the effect of Pax8 on the transcription of the NIS gene, thus confirming that these interactions are functionally relevant. Evidence has been accumulating concerning the role of CTCF in the establishment of intra-chromosomal loops which ultimately mediate protein-protein contacts between distal complexes and the general transcription machinery [49, 50]. On the other hand, Sp1 is a ubiquitously expressed transactivator, which physically interacts with several components of TFIIB and TFIID (mentioned above as potential Pax8 interacting proteins) and factors related to epigenetic events, such as histone deacetylases and p300/CBP histone acetyltransferase . Interestingly, several studies have described synergistic interactions between Pax8 and p300 acetyltransferase for enhancing the transcriptional activity of thyroid-related genes [52, 53]. Taking into account this complete transcriptional scenario, our data describe potential interactions of Pax8 with both common TFs and core elements, which could cooperate in chromatin remodeling for transcriptional regulation in thyroid cells.
Identification of biological processes controlled by Pax8 in thyroid cells
Pax8 has been mainly associated to thyroid differentiation and development through its transcriptional role in key thyroid-related genes [54, 55]. At this regard, we observed a downregulation of DIO1 after abolishing Pax8 (Additional file 5), which potentially binds to a critical region for selenocystein insertion in the DIO1 mRNA. Data were recently provided indicating that TSH tightly regulates DIO1 expression in thyroid cells through Pax8-dependent DIO1 mRNA stabilization (S.G. Leoni; unpublished observations). Moreover, gene expression profiling in normal versus malignant thyroid tissues demonstrated a downregulation of DIO1 and DIO2 , which could be linked to Pax8 loss during cancer progression.
Intriguingly, Pax8 modulates the expression of several genes involved in carcinogenesis and thyroid malignancies (phosphatidyl-inositol/insulin and MAPK pathways) and cell cycle processes (CDKN2B
CCNB1 and CCNB2, among others) (Additional file 11). These findings are in accordance with previous studies in which Pax8 expression was abolished in the differentiated thyroid cell line FRTL5 [20, 57]. Our data would also explain the biological mechanism underlying the partial decrease in thyrocyte proliferation in response to both IGF-I and TSH (main regulators of thyroid proliferation and differentiation) after both Nkx2.1 and Pax8 mRNA silencing .
DNA-related biological processes involved a plethora of functional categories (replication, repair and metabolism), highlighting the novel finding of Brca1-dependence on Pax8. In this regard, Shih et al described that BRCA1 and BRCA2 germline mutations were twice as common in individuals developing a second non-ovarian carcinoma, with follicular thyroid carcinoma being one of the most frequent secondary tumours . This finding can be of great relevance in the development of sporadic thyroid tumors, given that, as mentioned before, Pax8 expression is decreased or lost in thyroid tumours.
Recent reports have associated the transcription factors Pax2 and Pax5 with increased capabilities for cell motility and adhesion in human cancer [58, 59]. In parallel with these Pax-related functions, we observed significant expression changes of loci involved in cell motion/adhesion, notably the Pax8 effect on NCAM1 (neural cell adhesion molecule 1) transcription. NCAM1 and other components of adherens junctions, such as cadherins, have been described to be essential for maintaining cell polarity and epithelial integrity . Interestingly, Cadherin-16 (Cdh16/Ksp-cadherin) was recently proposed to play a TSH-regulated role in thyroid development , and its expression and promoter activity is controlled by Pax8 [20, 62]. We have not only confirmed transcriptional regulation of Cdh16 by PAX8, but also defined additional PAX8-dependent genes that could be essential for thyroid cell polarity (MYO5b and Rab17, among others). In this regard, germline mutations in MYO5b have been associated with disruption of epithelial cell polarity in MVID (MIM251850) . This role is exerted via its involvement in vesicle trafficking through direct interactions with Rab GTPase proteins, such as RAB11a and RAB8a. Further functional studies should be performed to evaluate potential Myo5b interactions with RAB17, another Rab GTPase protein involved in membrane trafficking and confirmed as a Pax8 target in the present study.