VALORIZACION DE LA CADENA TEXTIL PRODUCTIVA

With the ParaHox cluster being the sister cluster to the Hox, it was expected that it may also exclude transposable elements. This, however, was not seen to be the case, and three studies have now identified transposable elements throughout the amphioxus ParaHox cluster (Ferrier et al., 2005; Osborne and Ferrier, 2010; Osborne et al., 2006), despite conserved gene spacing and cluster organisation across the Chordata (Ferrier et al., 2005). This places the chordate ParaHox cluster in stark contrast to the Hox cluster of chordates (Osborne and Ferrier, 2010), where TEs seem to be actively excluded and pushed to the 5’ and 3’ of the chordate Hox clusters (Amemiya et al., 2008). The work here (section 3.3.9 and figures 3.12-3.14) further supports this and an abundance of additional TEs and TE fragments are seen spread throughout the B.floridae ParaHox cluster.

It is thought that the ability of TEs to invade the ParaHox cluster, unlike the Hox cluster, may be linked to the open state of the ParaHox cluster within the germline (Osborne and Ferrier, 2010), with the clustered mouse Cdx1 ParaHox gene shown to be transcriptionally active within germline cells (Kurimoto et al., 2008). This would then suggest additional selective pressures are involved in maintaining an intact ParaHox cluster within the chordates given the propensity of TEs to facilitate genomic rearrangement. One such constraint could be the presence of genomic regulatory blocks (GRBs) and long range enhancers maintaining the relative positions and organisation of genes (Kikuta et al., 2007). The state of TE content has not yet been examined within the recently

discovered intact ParaHox clusters of the echinoderms Patiria miniata and Acanthaster planci (Annunziata et al., 2013; Baughman et al., 2014) and hemichordate Ptychodera flava (Ikuta et al., 2013). It would be interesting to examine the TE content of these species to determine whether TEs are invading the ParaHox cluster throughout the Deuterostomia, or if this is a unique feature of the chordate phylum. One interesting aspect that has been made clearer from this analysis is that the density of TEs immediately surrounding the ParaHox cluster is much higher than that of within the ParaHox cluster, particularly surrounding PRHOXNB, where 28 TEs exist in the 40kb upstream of Cdx (figure 3.10). Of those elements that do exist within the ParaHox cluster, including the regions immediately upstream of Gsx and Cdx, a total of 10 TEs exist within the ParaHox cluster proper, and all bar two TEs exist in the intergenic region between Gsx and Xlox, something also seen in (Osborne et al., 2006), though the number of elements described here is less than the 16 described in this intergenic region within the ParaHox PAC sequence (Osborne and Ferrier, 2010). It would thus be

111 interesting to examine whether the Osborne et al TEs similarly localise to non-conserved regions. The remaining two elements, LanceleTn4 and LanceleTn3a, are located either side of Cdx exon2. Taken together, this suggests that there may be some constraint at work preventing TEs from

invading some regions of the ParaHox cluster, perhaps instead targeting them to regions less integral to the regulation of the ParaHox genes. Further work would be needed to analyse whether any constraints on the ability of TEs to invade the ParaHox cluster do indeed exist. Several intact ParaHox clusters are now available beyond those analysed in (Osborne and Ferrier, 2010). One could start by analysing the TE content of B.lanceolatum and B.belcheri to examine if the localisation of TEs across these clusters agree with that of B.floridae, as well as those of the aforementioned echinoderm and hemichordate clusters. The presence of TEs within the ParaHox cluster may also serve as a useful tool for the identification of regions with important regulatory function, as TEs are unlikely to invade such regions without harming the development of embryos. When combined with the other

analyses carried out in this work, such as the VISTA analysis in section 3.3.4 (figure 3.4), it may help inform the targeted screening for regions of regulatory importance to the ParaHox genes.

The high abundance of TEs within the intergenic region between Gsx and Xlox in

amphioxus poses the question as to whether this could serve a functional purpose. The Gypsy TE in Drosophila functions as an insulator, preventing distal enhancers from interacting with a promoter region, and other TEs have been shown to have epigenetic affects (reviewed in Slotkin and

Martienssen (2007)). This raises the question that perhaps the opening of intergenic regions within the ParaHox cluster to TE invasion may be intrinsically involved in their regulation. The presence of TEs between ParaHox genes would place them in regions where insulator elements might be expected. This is particularly notable in as TEs lie within the intergenic region between Gsx and Xlox, but not immediately upstream of ParaHox genes where presumptive promoter regions would lie, nor within intronic regions (bar a single LanceleTn element immediately next to Cdx exon 2). Of course, this is just speculation, but may be worth further investigation. Still, it does appear from this study, and by comparison with previous work by Osborne et al. and Ferrier (Ferrier et al., 2005; Osborne and Ferrier, 2010; Osborne et al., 2006) that TEs are excluded from the regions where promoter and enhancer elements may be expected, in the immediate upstream and intronic regions of the ParaHox genes, suggesting some constraint on TE invasion into the ParaHox cluster.

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Chapter 4. SCP1: An example of retrogene replacement adjacent to the amphioxus