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  • GLI proteins activate or repress

    2022-08-12

    GLI proteins activate or repress transcription of their target genes by binding to a similar sequence motif within a cis-regulatory module (CRM) (Hallikas and Taipale, 2006, Peterson et al., 2012). Transcriptional responses to the HH pathway can be elicited either by de-repression of GLI-R or in other cases, by transcriptional activation through GLI-A (reviewed in Falkenstein and Vokes, 2014). Recent studies suggest that additional tissue-specific factors are necessary for activating appropriate GLI target genes (Biehs et al., 2010). In the neural tube, GLI-bound CRMs are enriched for Sox binding motifs, and SOX2 and SOXB1 proteins act as neural-specific GLI co-factors (Oosterveen et al., 2012, Oosterveen et al., 2013, Peterson et al., 2012). The mechanisms underlying transcriptional specificity in other HH-mediated developmental processes remain poorly understood. In several contexts, CRMs associated with GLI-target genes that are closest to the Hh signaling source have higher affinity Gli binding sites, while genes farther away are associated with CRMs that contain lower affinity Gli AZD-4547 (Oosterveen et al., 2012, Parker et al., 2011, Peterson et al., 2012). In the vertebrate limb bud, Sonic hedgehog (Shh) signaling regulates digit number and growth (Chiang et al., 1996, Towers et al., 2008, Zhu et al., 2008). The timing and duration of SHH is important for establishing polarity within the limb bud (Li et al., 2014a, Li et al., 2014b, Zhulyn et al., 2014), and there is some evidence suggesting that cells retain a memory of their exposure to SHH (Harfe et al., 2004). In addition, studies have suggested that a relatively brief exposure to SHH specifies digit patterning, while longer exposures are needed AZD-4547 for subsequent growth and expansion (Towers et al., 2008, Zhu et al., 2008). Shh expression in the limb bud is maintained by FGF proteins secreted from the apical ectodermal ridge (AER). Shh signaling regulates the transcription of the BMP inhibitor, Gremlin 1 (Grem1) (Zuniga et al., 1999, Panman et al., 2006, Zuniga et al., 2012, Li et al., 2014a, Li et al., 2014b, Vokes et al., 2008). GREM1 inhibits localized BMP activity, thereby maintaining the apical ectodermal ridge (AER). Together, these interactions comprise a signaling loop between the mesoderm and the AER that regulates limb growth and digit number (Khokha et al., 2003, Laufer et al., 1994, Litingtung et al., 2002, Michos et al., 2004, Niswander et al., 1994, Te Welscher et al., 2002, Verheyden and Sun, 2008, Zuniga et al., 1999).
    Material and methods
    Results A previous study assigned GLI-bound CRMs within the mouse limb to genes responsive to SHH signaling on the basis of their proximity to identify putative direct GLI target genes. This approach incorporated both distance to a SHH responsive gene as well as the gene density of the locus (Vokes et al., 2008). The expression patterns of 199 of the 205 previously predicted GLI target genes (Vokes et al., 2008) were determined at E10.5 and E11.5 (Fig. 1A). Of the 199 genes analyzed, 96% (n=191) were detected in embryos at E10.5, and of these, 90% (n=171) were detected in the limb (13 of 19 genes not detected in the limb at E10.5 were detected at E11.5) (Supplementary material Table S1). We classified genes based on their expression pattern in the limb at E10.5 (Appendix A Supplementary material, Appendix A Supplementary material). To identify genes that might primarily be regulated by SHH, we restricted our analysis to genes that were predominately expressed in the posterior limb bud, a region responsive to SHH signaling as defined by the expression of pathway target genes Gli1 and Ptch1 (Fig. 1B) (Litingtung et al., 2002, Marigo et al., 1996; Te Welscher et al., 2002; Chiang et al., 2001; Ahn and Joyner 2004). Posteriorly expressed genes cluster into three broad domains (Fig. 1C–E). Category 1 contained 24 genes expressed in the posterior and posterior-distal limb bud (Fig. 1C). Genes in this category included the SHH pathway target genes Gli1, Ptch1, and Ptch2 (Chiang et al., 2001, Litingtung et al., 2002; Te Welscher et al., 2002; Marigo et al., 1996; Motoyama et al., 1998). Category 2 comprised 12 genes expressed in the central limb (Fig. 1D), and category 3 contained 9 genes that were expressed in the posterior-proximal limb (Fig. 1E). Lastly, we identified 14 genes expressed in multiple domains, where at least one expression domain was spatially located within the SHH-responsive region. Because these may have more complex forms of regulation we excluded these genes from further analysis (Appendix A Supplementary material, Appendix A Supplementary material). Altogether, we identified 45 genes that were expressed in three domains in the limb bud. The genes in category 1 were associated with more GBRs (average of 2.9 GBRs per gene) compared to Categories 2 and 3 (average of 1.5 and 1.9 GBRs per gene respectively). There were no significant differences in the distribution of GBRs relative to the transcriptional start site for the genes in categories 1–3 (data not shown).