Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

Mammalian Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, Japan.
Developmental Cell (Impact Factor: 9.71). 01/2009; 16(1):47-57. DOI: 10.1016/j.devcel.2008.11.011
Source: PubMed

ABSTRACT The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription.

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    • "FISH also identifies differences in chromatin condensation at the submegabase level, between genomic regions in the same cell (Yokota et al. 1997; Gilbert et al. 2004), for a given region during differentiation in vitro (Chambeyron and Bickmore 2004; Morey et al. 2007) and in vivo (Williamson et al. 2012; Patel et al. 2013), or between wild-type and mutant cells (Eskeland et al. 2010; Nolen et al. 2013). FISH has also been used to examine tissue-specific colocalization of long-range enhancers and their target genes (Amano et al. 2009; Williamson et al. 2012) Although visually compelling, FISH and live-cell imaging are restricted to viewpoints corresponding to the regions detected by the probes used (Dostie and Bickmore 2012), are low-throughput assays, and have limited spatial resolution, although superresolution microscopy is improving the latter (Markaki et al. 2012; Nora et al. 2012; Patel et al. 2013). In contrast, the chromosome conformation capture (3C) technique and its derivatives—including circular 3C (4C), 3C carbon copy (5C), and chromosome capture followed by high-throughput sequencing (Hi-C) (for review, see de Wit and de Laat 2012; Ethier et al. 2012)—offer a genome-wide perspective on genome organization . "
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    ABSTRACT: 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. © 2014 Williamson et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 12/2014; 28(24):2778-2791. DOI:10.1101/gad.251694.114 · 10.80 Impact Factor
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    • "In the non-expressing tissue, the distance between the ZRS and the Shh gene was similar for each allele, particularly at the closest interval (<0.2 μm). In the ZPA, the conformation of the wild-type allele (WT F-ZPA, Fig. 5E) showed significantly closer association between the ZRS and the Shh gene than in non-expressing cells, in agreement with a previous report (Amano et al., 2009). In the insertion allele, however, the association was not significantly different from that in non-expressing cells (Fig. 5E), suggesting that although the ZRS is active at close range to drive lacZ expression, the insertion element inhibited long-range interactions. "
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    ABSTRACT: 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.
    Development 04/2014; 141(8):1715-25. DOI:10.1242/dev.095430 · 6.46 Impact Factor
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    • "However, there are also limitations to a simple SHH-based model for digit specification, including the robustness of the process. The expression of Shh has been shown to fluctuate during limb bud development (Amano et al., 2009), and neither removal of one copy of Shh (Bénazet et al., 2009; Chiang et al., 1996), nor posterior implants of Shh expressing cells alter the digit pattern (Riddle et al., 1993). Theoretical considerations suggest that for a single morphogen-threshold-based mechanism even small changes in concentrations at the source would shift the position at which the pattern would emerge (Figure 2C) (Lander et al., 2009). "
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    ABSTRACT: The mechanism that controls digit formation has long intrigued developmental and theoretical biologists, and many different models and mechanisms have been proposed. Here we review models of limb development with a specific focus on digit and long bone formation. Decades of experiments have revealed the basic signalling circuits that control limb development, and recent advances in imaging and molecular technologies provide us with unprecedented spatial detail and a broader view on the regulatory networks. Computational approaches are important to integrate the available information into a consistent framework that will allow us to achieve a deeper level of understanding and that will help with the future planning and interpretation of complex experiments, paving the way to in silico genetics. Previous models of development had to be focused on very few, simple regulatory interactions. Algorithmic developments and increasing computing power now enable the generation and validation of increasingly realistic models that can be used to test old theories and uncover new mechanisms.
    Birth Defects Research Part C Embryo Today Reviews 03/2014; 102(1). DOI:10.1002/bdrc.21057 · 2.63 Impact Factor
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