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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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    • "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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    • "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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