Composition of a regulatory locus and the impact on phenotype and disease

It was assumed that most mutations/variations that cause human diseases would be due to changes in the coding regions of genes affecting the structure of proteins. This view has changed dramatically over recent years and it is now clear that the majority of mutations related to disease predisposition and even variability within the human population occur outside genes. The assumption is that these differences reside in regions of the DNA (known as regulatory domains or enhancers) that control protein production in the cell. Our research focusses on how changes in these regulatory domains can lead to congenital abnormalities. Although development of the human foetus is highly reliable, in approximately 1 in 100 cases deformities occur. Some of the most frequent abnormalities that occur affect the skeleton of the arms and legs and structure of the head and face. We have identified mutations in regulatory domains responsible for a spectrum of deformities in these features of the newborn, by using mammalian model systems such as the mouse and our goals are to investigate the mechanism by which this class of mutations causes congenital defects.

Genome wide association studies (GWAS) highlight SNPs (single nucleotide polymorphisms) in noncoding DNA associated with disease traits for complex or common diseases. Undoubtedly, cis-regulatory mutations cause multiple human congenital defects which show a direct association of regulatory mutations with effects on gene expression. Enhancer adoption and recently, ectopic enhancer activity were shown to occur in patients with chromosomal deletions or duplications that remove TAD boundaries. Hence, examination of the mechanisms by which regulatory mutations perturb gene expression is fundamental to understanding the basis of congenital defects.
We showed that disrupted gene regulation of the signalling molecule SHH is responsible for skeletal abnormalities and others have shown involvement in brain and craniofacial defects. Mutations in the Shh limb-specific cis-regulator, the ZRS, cause appendicular skeletal defects which has subsequently, led to the categorization of a broad spectrum of defects into the ‘ZRS associated syndromes’. Our studies range from defining causative mutations to attempting to identify regulatory processes involved in disease, focusing on causative mechanisms and investigating general principles that underlie long range gene activation. We showed that dominant regulatory mutations result from both gain and loss of transcription factor binding capacity. One series of point mutations in human hijacks the normal regulatory machinery involved in setting the Shh expression boundary to generate ectopic expression and skeletal defects.
In addition we are investigating the role that chromatin organization plays in supporting enhancer activity and controlling gene expression. In general, we are investigation the overall architectural components required for cis-regulators to operate over large genomic distances. Known chromatin organizers such as CTCF binding sites are being surveyed to understand their direct role on gene expression and their contribution to the phenotype of the developing mouse. Systematic removal of CTCF sites in the large Shh regulatory domain results in change in 3D chromatin organization and one site underlies the appearance of the holoprosencephaly (craniofacial and brain defects) spectrum in mouse. We are presently developing imaging techniques to map chromatin changes that occur during developmental gene activation and to observe the dynamics in live cells. Our pursuit of long-range regulatory mechanisms are important in understanding developmental gene activation, and the contribution of non-coding mutations to congenital abnormalities.