Profiling of conserved non-coding elements upstream of SHOX and functional characterisation of the SHOX cis-regulatory landscape

Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox ( SHOX ) gene, regulated by seven CNEs located downstream and upstream of SHOX , with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis -regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS and (iii) the chromatin architecture of the SHOX cis -regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis -regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis -regulatory domain spanning more than 1 Mb and harbouring putative new cis -regulatory elements.