Transcriptional repression needs to be rapidly reversible during embryonic development. This extends to the Hedgehog pathway, which primarily serves to counter GLI repression by processing GLI proteins into transcriptional activators. In investigating the mechanisms underlying GLI repression, we find that a subset of GLI binding regions, termed HH-responsive enhancers, specifically loses acetylation in the absence of HH signaling. These regions are highly enriched around HH target genes and primarily drive HH-specific transcriptional activity in the mouse limb bud. They also retain H3K27ac enrichment in limb buds devoid of GLI activator and repressor, indicating that their activity is primarily regulated by GLI repression. Furthermore, the Polycomb repression complex is not active at most of these regions, suggesting it is not a major mechanism of GLI repression. We propose a model for tissue-specific enhancer activity in which an HDAC-associated GLI repression complex regulates target genes by altering the acetylation status at enhancers.

The Hedgehog (HH) pathway regulates a spectrum of developmental processes through the transcriptional mediation of GLI proteins. GLI repressors control tissue patterning by preventing sub-threshold activation of HH target genes, presumably even before HH induction, while lack of GLI repression activates most targets. Despite GLI repression being central to HH regulation, it is unknown when it first becomes established in HH-responsive tissues. Here, we investigate whether GLI3 prevents precocious gene expression during limb development. Contrary to current dogma, we find that GLI3 is inert prior to HH signaling. While GLI3 binds to most targets, loss of Gli3 does not increase target gene expression, enhancer acetylation or accessibility, as it does post-HH signaling. Furthermore, GLI repression is established independently of HH signaling, but after its onset. Collectively, these surprising results challenge current GLI pre-patterning models and demonstrate that GLI repression is not a default state for the HH pathway.

1Transcriptional repression needs to be rapidly reversible during embryonic 2 development. This extends to the Hedgehog pathway, which primarily serves to counter 3 GLI repression by processing GLI proteins into transcriptional activators. In investigating 4 the mechanisms underlying GLI repression, we find that a subset of these regions, 5termed HH-responsive enhancers, specifically loses acetylation in the absence of HH 6 signaling. These regions are highly enriched around HH target genes and primarily drive 7 HH-specific limb activity. They also retain H3K27ac enrichment in limb buds devoid of 8 GLI activator and repressor, indicating that their activity is primarily regulated by GLI 9repression. The Polycomb repression complex is not active at most of these regions, 10suggesting it is not a major mechanism of GLI repression. We propose a model for 11 tissue-specific enhancer activity in which an HDAC-associated GLI repression complex 12 regulates target gene expression by altering the acetylation status at enhancers. 13 14 131 HH-responsive GBRs are distal enhancers containing high quality GLI motifs 132Although Stable GBRs are not highly enriched at HH target genes, 62% of them are 133 located in close proximity to the promoters of genes, compared to 26% of HH-134

The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We found that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we found that Gli1 and Nanog are co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway.The HH 3 pathway is essential for regulating biological processes in a diverse set of cells. HH ligands, including Sonic hedgehog, bind to the Patched1 (PTCH1) receptor, which activates the transmembrane protein Smoothened, resulting in pathway activation (for a review, see Ref. 1). The transcriptional response to HH ligands is mediated by the GLI family of transcription factors (GLI1-3), which can act as both transcriptional activators and repressors in a context-dependent fashion (for a review, see Ref. 2). The presence of HH ligand causes the maturation of full-length GLI proteins into their transcriptional activator forms (GLI-A), whereas in the absence of ligand GLI proteins undergo C-terminal truncation and then act as transcriptional repressors (GLI-R). Although GLI2 and GLI3 exist in both activator and repressor forms, GLI2 is the major activator, whereas GLI3 is the major repressor (3-5). In contrast, GLI1 only exists as a full-length activator form (5-7). Gli1 is a direct HH target gene, thereby participating in a positive feedback loop (8 -11).The HH pathway was initially characterized for its role in regulating embryonic development, but it also has critical roles in regulating the homeostasis of several adult tissues (for a review, see Ref. 12). In particular, HH regulates two major neural stem cell populations in the brain, the ventral subventricular zone and subgerminal zone, as well as quiescent hair follicle stem cells (13). In the absence or inhibition of the HH pathway, these tissues undergo a marked reduction in the number of proliferating cells, indicating that the pathway is required for normal proliferation (14). Conversely, hyperactivation of the HH pathway results in an expanded population of neural stem cells. In this context, the progeny of neural stem cells is shifted so that they preferentially give rise to two daughter stem cells instead of producing transient amplifying cells capable of generating differentiated progeny (15). Together these results indicate that the levels of HH perceived by neural stem cells regulate the balance between generating stem cells and differentiated progenitors. In addition to regulating normal neural development, various studies have suggested that populations...

The Hedgehog (HH) signaling pathway is primarily modulated by GLI transcriptional repression in the mouse limb. Previous studies have suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. Despite this, we find that SMARCC1 maintains chromatin accessibility at GLI enhancers suggesting that it contributes to enhancer activation rather than helping to mediate GLI repression. Furthermore, SMARCC1 binds GLI-regulated enhancers independently of GLI3 and does not facilitate transcriptional repression of most GLI target genes. Finally, Smarcc1- and Shh- double knockout phenotypes suggest that SMARCC1 is not required to mediate constitutive GLI repression in HH mutants. We conclude that the BAF complex does not mediate GLI3 repression, which we propose instead utilizes alternative chromatin remodeling complexes.