Thus, if a risk SNP is localized to a putative enhancer element defined through chromatin profiling, it is essential that the putative enhancer is functionally validated.
Rs9485370 is located in a putative enhancer element in HMEC cell lines and is linked to a number of SNPs which have been predicted to influence transcription factor binding.
One SNP (rs4431561, r with rs4242382 = 0.949) fell within a highly conserved region, one SNP (rs1447296, r with rs4242382 = 0.952) was located in a putative enhancer region, and one SNP (rs7812894, r with rs4242382 = 1.0) was located in a predicted region of regulatory potential.
This is also suggested by the stomata clustering phenotype that is observed in leaves of a putative enhancer trap line of AP2C3::AP2C3-GFP where AP2C3 protein levels were increased within its own expression domain (data not shown).
Taken together, the phenotypic and expression data point to a role for CALS5 in pollen mother cells and pollen grains, and, although no vegetative phenotypes were observed, expression in vegetative tissues is also possible, due to a putative enhancer more than 1.8 kb upstream of the coding sequence.
Additionally, there is a putative exonic splicing enhancer at the beginning of locus 2 (nt 1475 to 1482).
For example, in the LMP-2B gene, locus 1 overlaps a splice donor site (after nt 788, Additional file 3), which is buried within the stem of a tetraloop hairpin; towards the 3′ end of this structure (nt 827 to 833) is a putative intronic splicing enhancer.
We also tested whether DNase I would be more effective as a discriminator than H3K27ac ChIP-seq for predicting whether a putative enhancer will fail to be expressed in the brain.
In addition, these sequences with a putative enhancer activity have not been tested in their natural context, which can be represented by clusters of several overlapping motifs with complex and often antagonistic interactions as already demonstrated for the CFTR gene [ 29].
Therefore, it was used as a positive control in the experiments on the identification of a putative enhancer within the D4Z4.
