Techniques and assays to study enhancer activity and functions

1.2.4.1 ChIP-chip and ChIP-seq

The methods of ChIP-chip and ChIP-seq have allowed the identification of enhancers on a genomic scale through detecting the binding of chromatin marks and cofactors on the regulatory sequences. These methods are based on traditional ChIP experiments with different downstream analysis. Briefly, ChIP involves crosslinking of DNA- binding proteins with the DNA by treating cells with formaldehyde and shearing chromatin usually by sonication. An immunoprecipitation of the cross-linked chromatin is performed using an antibody against the specific TF or protein of interest. This results in the identification of all the binding sites in the genome for the protein of interest. After reversal of the crosslink and purification of the precipitated chromatin fragments the sample can be analysed by PCR to study particular gene. However, genome-wide analysis can be performed by microarray (ChIP-chip) or sequencing (ChIP-seq). For ChIP-chip the immunoprecipitated sample and the input chromatin, as a control, are labeled with fluorescent dyes and are hybridized to microarrays. For the ChIP-seq instead the immunoprecipitated DNA fragments are sequenced through next-generation sequencing techniques, and computationally mapped to a reference genome. The results from ChIP-seq are based on statistical analysis of read counts and advanced computational ChIP-seq analysis tools are available to identify ChIP-seq peaks. The ChIP-seq analysis covers the entire mappable portion of the reference genome without need to restrict the analysis to its subregions, as it happens instead in the ChIP-chip by hybridization to microarrays. Mainly this improvement has contributed to the success of

the ChIP-seq as method of choice compared with the ChIP-chip, together with lower cost of the experiments and an unambiguous identification of the peaks (Farnham P. J., 2009, Visel et al., 2009b).

Although techniques like the ChIP-chip and ChIP-seq are very successfully for the identification of enhancer elements genome-wide, they make use of indirect properties of the regulatory elements (such as the occurrence of chromatin marks or binding of specific TFs of interest) rather than assessing their functionality and activity.

1.2.4.2 Transgenic gene reporter assays in vivo and in vitro

Transgenic reporter assays provide a more functional approach to test enhancers, by addressing their ability to drive gene expression. In a typical transgenic reporter assay a putative enhancer fragment is cloned upstream of a reporter gene driven by a promoter, which has a minimal or no activity by itself, but that respond to the input of the adjacent enhancer. In this way the activity of the enhancer is revealed by the expression of the reporter gene.

In vivo transgenic mouse reporter assays are one of the most used techniques to detect

enhancer activity in vivo. In these experiments the enhancer to test is linked to the reporter gene, typically LacZ, and then the transgene construct is delivered into mouse zygotes through pronuclear injection. The resulting transgenic embryo will be tested for ß-gal activity to visualise the expression pattern and in vivo activity of the enhancer element in the embryonic tissues.

In vivo transgenic mouse assay can’t be used for quantitative analysis of the enhancer

activity nor to detect modest alteration to enhancer intensity or quantitative effects of enhancer mutations. These effects and a quantitative measure of enhancer activity have

been studied predominantly using in vitro reporter assays, where the enhancer is coupled to a luciferase reporter gene and transiently transfected into cells. The transgenic reporter gene intensity can be measured quantitatively with the use of a luminometer (Rosenthal, 1987, Naylor, 1999, Schenborn and Groskreutz, 1999).

The luciferase assay can allow quantification of the gene expression driven by an enhancer element in different cell lines for instance to test the specificity of the enhancer in different cell context that might be similar or very different according to the choice of the experimental systems and the purposes of the comparison. In the same way, the effect of the input of specific TFs activity on the enhancer can be measured by gain or loss of function of the TF of interest or by manipulating the amount of the same TF. Finally, effects of mutations in the enhancer sequence, for instance in the consensus motifs of known TFBSs, can be detected and quantified. For these applications, the luciferase assay persists as one of the best techniques to study and assess the biological function of enhancers and to characterise the functions of the several modules that makes the enhancers and the effect of specific TFs recognising and binding to the TFBSs. Despite the advances of large-scale genome-wide techniques to identify and characterise enhancers in vivo, luciferase assay experiments are still needed in parallel to these approaches to understand the biological function of enhancers, and the mechanisms of their activation and regulation by TFs. The work presented in this thesis demonstrates the advantage of the application of this technique.

1.2.4.3 Chromosome Conformation Capture (3C) assay

A big remaining challenge in the study of enhancers is to determine the relationship between enhancers and their target genes. Although comparisons between ChIP-chip or ChIP-seq with transcriptome data from microarray and RNA-seq can give clues in the

association enhancer-target gene, they can’t give evidence of a direct interaction enhancer-promoter and therefore enhancer-target gene which would be necessary in the establishment of gene regulatory networks on a genomic scale. Current views support the idea that most enhancers establish direct physical interactions with their target gene promoters (Symmons and Spitz, 2013). These interactions can be detected by chromosome conformation capture assay (3C) and its derivative technologies. This technique is based on a biochemical strategy that allows representation of the 3D organisation of the DNA and the chromosome topology. Subsequent steps of fixation, digestion, and re-ligation of fixed chromatin followed by quantification of the ligation junctions allows obtaining a one dimension cast of the 3D chromatin structure (Zhu et al., 2013, de Wit and de Laat, 2012). Overall, the main picture emerging from studies based on these approaches is that both promoters and enhancers are frequently engaged in multiple interactions so that enhancer-promoter interactions are not exclusive. For most genes, the elements that regulate their expression will be found in cis, although at distances that could be hundreds of kilobases. However, enhancers have been reported to act also promiscuously, activating neighbouring but biologically irrelevant genes (Symmons and Spitz, 2013). In conclusion, these studies are pointing out that many current approaches taken to associate enhancers with their target genes, such as the previously mentioned comparison of ChIP-seq data with transcriptome data, might be misleading and new approaches need to be developed.

1.2.5 Gene Regulatory Networks in Embryonic Stem Cells and Neural