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Via bioinformatic analysis and gene expression profiles, the list of transcription factors predicted

to bind the ZIC2 3’UTR was narrowed down to nine candidates, including ZIC2. All nine of the

identified candidates are known to be required for correct embryo development, with a particular focus on gastrulation. Additionally, four of the nine candidates’ binding sites are predicted to be lost when SNVs are introduced (CDX2, SOX2, SOX3 and LHX1), whilst four binding sites are predicted to be created upon SNV introduction (FOXA2, FOXJ1, SOX2 and SOX3). This

suggests that SNVs have the potential to markedly disrupt ZIC2 NCE activity by changing the

nature of enhanceosome or repressosome formation. Further in vitro studies are required to

determine if these TF candidates do indeed interact with the ZIC2 NCE to regulate transcription,

and how the SNVs affect these interactions (see Chapter 5).

One candidate TF, CDX2, is known to play an important role in somitogenesis, endoderm

development and neural tube closure during embryonic development (Savory et al., 2011), and

is required at gastrulation for AP patterning and tissue extension via the regulation of Hox genes

and Wnt signalling (Young et al., 2009). This regulation of Hox genes occurs via the binding of

CDX2 to two Hoxc8 enhancers, a gene critical for early development of the neural tube and

two enhancers prevented CDX2 from binding specifically to both sites and resulted in altered

transgene expression in the neural tube and somites of developing embryos (Taylor et al., 1997).

Similarly, the ability to bind an enhancer element has been shown for LHX1 (Costello et al.,

2015), FOXA2 (Gao et al., 2008) and SOX2 (Yuan et al., 1995). Moreover, LHX1 and FOXA2 have

been shown to complex, together with OTX2 and LDB1, to regulate target gene expression and

direct anterior mesendoderm, node and midline development (Costello et al., 2015). Together,

these studies provide precedent for the identified candidate TFs to actively bind ER elements

such as the putative ZIC2 NCE.

Whilst candidate transcription factors were analysed for their expression patterns at

gastrulation and the occurrence of binding sites in the ZIC2 3’UTR, the list of factors identified

in this chapter is far from exhaustive. Information about the expression pattern at gastrulation of a majority of the TFs identified in the bioinformatics screen could not be found. Likewise, a majority of the 79 identified TFs with overlapping gastrulation expression patterns have not had

any known binding motifs published and therefore their ZIC2 3’UTR binding potential is

unknown. Whilst this study focused on genes that met both bioinformatic and expression criteria, it does not mean that identification of candidates should be restricted to the genes listed in Section 4.2.4. Analysis of enhancers based on predicted TFBSs alone is often dependant on knowledge of the preferred binding site of the candidate proteins. The binding preferences of many candidates are unknown at this stage and the difficulty of predicting candidates is further compounded by the fact that many TF require a co-factor to bind DNA (reviewed in Andersson 2014). Furthermore, whilst many predicted TFBSs have been shown to bind their target DNA in vitro, these results often do not translate to in vivo studies as they do not take into account variables such as cell type specificity and chromatin accessibility (Andersson, 2014). Similarly, it is important to note when analyzing binding site that short motifs frequently match to genomic or even random DNA sequences (for example, each 6 bp long motif would be

expected to occur every 46 bp = 4,096 bp), and only a small proportion of all matches in a

genome are typically bound by the corresponding transcription factor in vivo. Of these, only a

small number result in regulation of gene expression(Shlyueva et al., 2014; Yáñez-Cuna et al.,

2012). Thus, a large number of the candidate binding sites identified in this chapter are likely false-positives. Corresponding binding site predictions to gene expression patterns will remove a large number of these false positives, however there remains a possibilitiy that the final nine

candidate transcription factors may not interact with the ZIC2 NCE.

Together, this suggests that there remain multiple candidates yet to be identified. Thus, whilst

it is important to investigate the role of the nine identified candidates in regulating the ZIC2 NCE

Due to the nature of cis-acting NCEs, it is likely that the TFs that do bind the ZIC2 3’UTR are located nearby in the genome and that they serve a developmentally important role. As more information is acquired on gene expression patterns and binding sites are further clarified, the identification of new candidate TFs will become easier.

At this stage, FOXA2 is the only final candidate implicated in HPE development (Houtmeyers et

al., 2016). This affords an opportunity to identify a new selection of HPE related genes, as it is

likely that the introduction of the six SNVs will alter the interactions between the final nine

candidates and the ZIC2 NCE, resulting in congenital malformations.

4.3.3 Conclusion

The identification of mutations within a putative NCE in ZIC2 3’UTR in HPE probands by Roessler

et al (2012a) lead to the hypothesis that this element acts as an enhancer during gastrulation to

regulate ZIC2 transcription. In this Chapter I have shown that the ZIC2 NCE contains the

hallmarks of an active ER element, and have identified nine TF candidates to bind and regulate this element during gastrulation. Furthermore, I have shown that each of the six HPE-associated SNVs have the potential to disrupt the protein complexes that interact with the NCE. Though NCE function remains to be determined experimentally, it is likely an important component in