It is possible that even the non-conserved elements may contain a short sequence of conservation that is responsible for enhancing activities, particularly since the typical transcription factor binding site is just a few nucleotides-long. Interestingly, the two non-conserved enhancers, separated by only 57 kb, displayed the same pattern of reporter expression in the trigeminal ganglion. They could represent ? shadow enhancers ? with overlapping activities, but it remains unknown whether the target gene of these enhancers is Olig or a more distant or unannotated gene. Since we screened a BAC mapped within an orthologous fragment studied in the ENCODE project pilot phase, we asked whether our identified conserved enhancer 5F7 carried annotations suggestive of function. Human 5F7 does not show any significant DNaseI Paclitaxel hypesensitivity in the seven cell lines tested. Interestingly, human 5F7 is mostly covered by repressive chromatin marks in all cell lines investigated by ENCODE. However, the most conserved part of human 5F7 is marked by monomethylation on lysine 4 of histone H3 in embryonic stem cells, a modification associated with enhancers. This suggests that the locus is tightly regulated and mostly repressed but can be activated in a specific spatiotemporal manner. Such a tight control pattern would be compatible with the GDC-0879 likely regulation of OLIG genes. These data should be treated with caution however as they originate from non-neural human cell lines that likely differ in their regulation of this locus compared to LacZ positive cells in our E11 murine embryos. We also looked at p300 binding sites in forebrain, midbrain and limbs of E11 mouse embryos, but none of our identified enhancers overlapped with a peak of p300 binding in these tissues. The ENCODE project pilot phase had previously described several functional regions that showed no evidence of evolutionary constraint. Likewise, another report had subsequently suggested that non-conserved elements could also harbour enhancer activities in zebrafish transgenics, but a broad unbiased screen had not so far been conducted in mice. Here, we provide further evidence that non-conserved sequences with enhancer activity exist. This observation has important implications regarding the annotation of genomes and the identification of disease-related variation. It is noteworthy that our study presented two limitations precluding the exhaustive identification of enhancers in the DNA region under study.