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: 10.37). 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 · 12.64 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 · 3.87 Impact Factor
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    • "It is also possible that many noncoding regulatory mutations to date have not been identified simply because often only coding sequence is searched for mutations. For either or both of these reasons, Mendelian effects of mutations in cis-regulatory elements are exceedingly rare, the most prominent example to date perhaps being the Shh limb enhancer located within an intron of the Lmbr1 gene (Amano et al., 2009). This enhancer is, as far as is known, nonredundant, and thus perhaps more vulnerable to mutation or deletion (Sagai et al., 2005). "
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    ABSTRACT: Whole exome sequencing and, to a lesser extent, genome-wide association studies, have provided unprecedented advances in identifying genes and candidate genomic regions involved in the development of human disease. Further progress will come from sequencing the entire genome of multiple patients and normal controls to evaluate overall mutational burden and disease risk. A major challenge will be the interpretation of the resulting data and distinguishing true pathogenic mutations from rare benign variants.While in model organisms such as the zebrafish,mutants are sought that disrupt the function of individual genes, human mutations that cause, or are associated with, the development of disease, are often not acting in a Mendelian fashion, are frequently of small effect size, are late onset, and may reside in noncoding parts of the genome. The zebrafish model is uniquely poised for understanding human coding- and noncoding variants because of its sequenced genome, a large body of knowledge on gene expression and function, rapid generation time, and easy access to embryos. A critical advantage is the ease of zebrafish transgenesis, both for the testing of human regulatory DNA driving expression of fluorescent reporter proteins, and the expression of mutated disease-associated human proteins in specific neurons to rapidly model aspects of neurological disorders. The zebrafish affords progress both through its model genome and it is rapidly developing transparent model vertebrate embryo.
    Developmental Neurobiology 03/2012; 72(3):415-28. DOI:10.1002/dneu.20888 · 4.19 Impact Factor
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