Iain Williamson

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly

Summary Sonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

SUMMARY In humans, nearly two meters of genomic material must be folded to fit inside each micrometer-scale cell nucleus while remaining accessible for gene transcription, DNA replication, and DNA repair. This fact highlights the need for mechanisms governing genome organization during any activity and to maintain the physical organization of chromosomes at all times. Insight into the functions and three-dimensional structures of genomes comes mostly from the application of visual techniques such as fluorescence in situ hybridization (FISH) and molecular approaches including chromosome conformation capture (3C) technologies. Recent developments in both types of approaches now offer the possibility of exploring the folded state of an entire genome and maybe even the identification of how complex molecular machines govern its shape. In this review, we present key methodologies used to study genome organization and discuss what they reveal about chromosome conformation as it relates to transcription regulation across genomic scales in mammals.

Anterior-posterior differences in HoxD chromatin topology in limb development

A late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5′ Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5′ HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5′ HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb).

Development of five digits is controlled by a bipartite long-range cis-regulator

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.

Enhancers: From Developmental Genetics to the Genetics of Common Human Disease

In mammals, long-range gene regulation became apparent through simple Mendelian disease genetics in human and developmental genetics in the mouse. Can the insights into gene control, provided by the study of these enhancers, help us understand the functional significance of sequence variation associated with common/complex human disease and quantitative traits?

Shh and ZRS enhancer co-localisation is specific to the zone of polarizing activity

Limb-specific Shh expression is regulated by the (∼1 Mb distant) ZRS enhancer. In the mouse, limb bud-restricted spatiotemporal Shh expression occurs from ∼E10 to E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS colocalisation only in the Shh-expressing regions of the limb bud, in a conformation consistent with enhancer-promoter loop formation. However, in all tissues and at all developmental stages analysed, Shh-ZRS spatial distances were still consistently shorter than those to a neural enhancer located between Shh and ZRS in the genome. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS colocalisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh and ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate the expression of Shh. Summary: Super-resolution microscopy reveals that, during mouse limb development, enhancer-driven gene expression results in the juxtaposition of Shh and its limb bud-specific enhancer only within cells of the distal posterior limb bud.

PARP mediated chromatin unfolding is coupled to long-range enhancer activation

Enhancers are critical regulators of gene expression and can be located far from their target gene. It is widely assumed that mechanisms of enhancer action involve reorganization of three-dimensional chromatin architecture, but this is poorly understood. Here we identify a novel mechanism of long-range enhancer associated chromatin reorganization. At the Sonic hedgehog (Shh) locus we observe large-scale decompaction of chromatin between Shh and its brain enhancers in neural progenitor cells. We show that the chromatin unfolding is dependent on activation of the enhancers, not the promoter, is impeded by chromatin-bound proteins located between the enhancer and promoter, and is mediated by the recruitment of Poly (ADP-Ribose) Polymerase 1. We suggest that large-scale chromatin decompaction, analogous to the inducible puffs in Drosophila polytene chromosomes, represents a new mechanism of chromatin reorganization coupled to long-range gene activation from mammalian enhancers and that seems incompatible with a chromatin-looping model of enhancer-promoter communication

Polycomb-mediated chromatin compaction weathers the STORM

A recent super-resolution imaging study by Boettiger et al. elegantly demonstrates that three epigenetically defined, and functionally disparate, chromatin states have distinct folding characteristics in Drosophila nuclei.

06-P035 Additional PEA3 binding sites in the long range limb regulator disrupt posterior restriction of SHH causing polydactyly

Idiopathic Congenital Talipes Equinovarus, or clubfoot, is a common developmental disorder of the foot, affecting at least 2 in every 1000 live births in Scotland. The defect is characterised by a twisting of the foot and loss of calf muscle. Diagnosis is usually made on postnatal examination and treatment; usually a series of foot manipulations, takes place within the first year. Treatment can be particularly painful and is not always successful as the defect can recur leading to life-long disability in some cases. Very little is known of the aetiology of clubfoot despite it being such a common problem and therefore our lab set out to elucidate the developmental processes that occur during limb development which result in clubfoot. The chick limb is a well established model system used to study many developmental processes, signalling events and has been used to model several human limb disorders. We have developed a chick model of human clubfoot using a neuromuscular paralysing agent, resulting in a range of clubfoot conditions. Our lab is currently using this chick model to determine the developmental basis of the disorder. Initial phenotypic analyses of the chick clubfoot limb correlate well with those observed in human clubfoot; both exhibit muscle loss, tendon problems and bony abnormalities. We will use this chick model of human clubfoot to test our hypothesis that clubfoot may result from a failure of hindlimb rotation and elucidate the developmental and molecular basis of this congenital defect.