MRC Human Genetics Unit
Medical Research Council Human Genetics Unit

Robert Hill: Chromatin Organisation and the Regulation of Gene Expression

Research Programme

Summary

Birth defects occur as errors in human development and appear as alterations in body form in the foetus. Of the genetic factors involved, it is estimated that there are well over 1700 different inherited human disorders that alter embryogenesis with the majority of these causing multiple defects. An understanding of the basis of these birth defects requires an investigation into the nature of the genes responsible; however, it has been recognised more recently that mutations not only in genes but also in regulatory elements, such as enhancers, are responsible for congenital abnormalities. Genes that control development are often regulated by a series of long range acting enhancers and our work has revealed a paradigm for long-range gene regulation which focuses on the sonic hedgehog (Shh) gene. Our goals are two-fold: 1.) to understand the Shh signalling pathway that organizes vertebrate organogenesis and 2.) to identify processes involved in developmental gene expression by investigating the spatial organisation of long range regulatory loci, the DNA topology that activates and constrains the locus and the gene network (and epigenetic modifications) that operates at enhancers to activate gene transcription.

Approaches

Genes that encode crucial developmental factors must be expressed faithfully in the correct cells of the embryos and at the right time of development. Mutations that cause misexpression of potent developmental regulators provide unique pathogenetic mechanisms. Understanding these mechanisms will lead to both insights into the disease process and a better grasp of the normal regulatory machinery. We use approaches that combine mouse molecular genetics, chromosomal engineering, transcriptome analysis and biochemistry to gain insights into the signalling and transcriptional networks that orchestrate vertebrate organogenesis.

Projects include:

  1. Precision chromosomal engineering using transposons to structurally manipulate the long range domain of the Shh regulatory locus.
  2. Chromatin conformation technologies to define components that compose the DNA topology necessary for long range gene regulation
  3. CRISPR/Cas9 mutagenesis to make subtle mutations to define structural elements within the Shh regulatory domain
  4. Epigenetic analysis of the enhancer accompanying gene activation
  5. Protein/DNA interactomics to define the gene network involved in gene activation that specifies the organising center (the zone of polarising activity) of the limb bud
B.Hill research figure 1
Figure 1. Long Distance Regulatory Domain of Shh

Purpose

We investigate the embryonic mechanisms that organise the overall design of the developing organ systems. These mechanisms define the structures and their final arrangement in composing the embryonic anatomy. Our aims are to investigate developmental and gene regulatory processes that participate in organogenesis and understand how genetic inaccuracies lead to human congenital abnormalities. We aim to identify the transcriptional network of genes that specify cellular identity in the well-established and highly accessible genetic system of the developing limb bud and how these organise the development of the skeleton. The relationship of enhancer structure to function is poorly understood and our work aims to understand how enhancers are activated during development, and once activated, how enhancers situated a long distances from their target promoters (up to a million bases away) conveys this information to regulate accurate temporal and spatial gene expression.

Background

Regulation of Shh gene expression

Sonic hedgehog (Shh) is a key signalling molecule required for normal development of many of the organ systems in the embryo including head and face, brain, neural tube, lung, gut and skeleton.  Within these tissues the developmental role that Shh plays is dependent on complex gene regulation and like many developmental genes requires long-range regulatory mechanisms for its full spatiotemporal pattern of expression. With the Shh coding region lying adjacent to a large gene desert, the expression is controlled by a group of cis-regulators that lie upstream of the gene with the regulatory domain spanning nearly a million base pairs and which together control the complex gene expression pattern seen in the embryo.

B.Hill research figure 2
Figure2. Preaxial Polydactyly Type2 Skeletal Abnormalities

Limb Abnormalities

Mutations of the SHH gene causes holoprosencephaly; whereas, inappropriate activation of the SHH signalling pathway causes medulloblastoma, pancreatic neoplasia, basal cell carcinoma and skeletal defects classified as preaxial polydactyly type 2 (PPD). PPD is a result of mutations in a cis-regulatory element called the ZRS which controls the expression of Shh in the developing limb bud (Fig. 2). The ZRS is an extreme example of a long-range regulator. In the human genome, the ZRS resides at a distance of 1Mb from the SHH gene and is located within the intron of another gene that itself has no role in limb development. The data are compelling that PPD is a regulatory disorder resulting from mutations within the ZRS that causes misexpression in the limb bud.