KRAS Promotes GLI2-Dependent Transcription during Pancreatic Carcinogenesis.

Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Circular and Fusion RNAs in Medulloblastoma Development.

Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5' to 3' with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated.

Circular RNAs in Hedgehog Signaling Activation and Hedgehog-Mediated Medulloblastoma Tumors.

Within the past decade, circular RNAs have largely emerged as novel regulators of human biology, including brain function and cancer development. On the other hand, the Hedgehog pathway has established roles in regulating biological processes, including tumorigenesis. Here, the circular RNA transcriptome, in the context of Hedgehog signaling activation of medulloblastoma Daoy and human embryonic palatal mesenchyme HEPM cells, was determined. In total, 29 out of the 30 selected circular RNAs were validated by Sanger sequencing, with some regulated to a limited extent by Hedgehog signaling. Interestingly, back-spliced junctions, the marker of exonic RNA circles, were also identified at a low frequency within poly (A) mRNAs, reflecting exon repetition events. Thirteen circular RNAs had reduced expression in human medulloblastoma tumors in comparison to normal cerebellum. For seven out of these thirteen RNA circles, the linear mRNAs originating from the same genes did not exhibit a reduced expression. Depletion and/or overexpression of these seven circular RNAs minimally affected medulloblastoma cell proliferation. These findings highlight that differential expression of a gene product may not necessarily elicit an obvious phenotypic impact. Consequently, further analysis is required to determine the possible subtle contributions to the development of this cerebellar tumor.

RITA downregulates Hedgehog-GLI in medulloblastoma and rhabdomyosarcoma via JNK-dependent but p53-independent mechanism.

Overactivation of the Hedgehog (HH) signaling pathway is implicated in many cancers. In this study, we demonstrate that the small molecule RITA, a p53 activator, effectively downregulates HH signaling in human medulloblastoma and rhabdomyosarcoma cells irrespective of p53. This is mediated by a ROS-independent activation of the MAP kinase JNK. We also show that in vitro RITA sensitized cells to the GLI antagonist GANT61, as co-administration of the two drugs had more pronounced effects on cell proliferation and apoptosis. In vivo administration of RITA or GANT61 suppressed rhabdomyosarcoma xenograft growth in nude mice; however, co-administration did not further enhance tumor suppression, even though cell proliferation was decreased. RITA was more potent than GANT61 in downregulating HH target gene expression; surprisingly, this suppressive effect was almost completely eliminated when the two drugs were administered together. Notably, RNA-seq demonstrated a broader response of pathways involved in cancer cell growth in the combination treatment, providing a plausible interpretation for tumor reduction in the absence of HH signaling downregulation.

Identification of novel GLI1 target genes and regulatory circuits in human cancer cells.

Hedgehog (HH) signaling is involved in many physiological processes, and pathway deregulation can result in a wide range of malignancies. Glioma-associated oncogene 1 (GLI1) is a transcription factor and a terminal effector of the HH cascade. Despite its crucial role in tumorigenesis, our understanding of the GLI1 cellular targets is quite limited. In this study, we identified multiple new GLI1 target genes using a combination of different genomic surveys and then subjected them to in-depth validation in human cancer cell lines. We were able to validate >90% of the new targets, which were enriched in functions involved in neurogenesis and regulation of transcription, in at least one type of follow-up experiment. Strikingly, we found that RNA editing of GLI1 can modulate effects on the targets. Furthermore, one of the top targets, FOXS1, a gene encoding a transcription factor previously implicated in nervous system development, was shown to act in a negative feedback loop limiting the cellular effects of GLI1 in medulloblastoma and rhabdomyosarcoma cells. Moreover, FOXS1 is both highly expressed and positively correlated with GLI1 in medulloblastoma samples of the Sonic HH subgroup, further arguing for the existence of FOXS1/GLI1 interplay in human tumors. Consistently, high FOXS1 expression predicts longer relapse-free survival in breast cancer. Overall, our findings open multiple new avenues in HH signaling pathway research and have potential for translational implications.

Blockade of the Hedgehog pathway downregulates estrogen receptor alpha signaling in breast cancer cells.

Anti-estrogen treatment, exemplified by tamoxifen, is a well-established adjuvant therapy for estrogen receptor alpha (ERα)-positive breast cancer. However, the effectiveness of this drug is limited due to the development of resistance. The Hedgehog (HH) signaling pathway is critical in embryonic development, and aberrant activation of this transduction cascade is linked to various malignancies. However, it remains unclear whether HH signaling is activated in human breast cancer and related to tamoxifen resistance. Deciphering how this pathway may be involved in breast cancer is a crucial step towards the establishment of targeted combinatorial treatments for this disease. Here, we show that the expression of the HH signaling effector protein GLI1 is higher in tamoxifen resistant compared to sensitive cells. Tamoxifen resistant cells have stronger ERα transcriptional activity relative to sensitive cells, even though the ERα expression is similar in both cell types. Knockdown of GLI1 attenuates cell proliferation and reduces ERα transcriptional activity in both sensitive and resistant cells, irrespective of estrogen stimulation. Combinatorial treatment of tamoxifen and the GLI antagonist GANT61 further suppresses the growth of sensitive and resistant cells relative to administration of only tamoxifen, and this was irrespective of estrogen stimulation. Moreover, a positive correlation between GLI1 and ERα expression was identified in breast cancer samples. Additionally, high GLI1 expression predicted worse distant metastasis-free survival in breast cancer patients. These data suggest that the HH pathway may be a new candidate for therapeutic targeting and prognosis in ERα-positive breast cancer.

Tamoxifen Resistance: Emerging Molecular Targets.

17ß-Estradiol (E2) plays a pivotal role in the development and progression of breast cancer. As a result, blockade of the E2 signal through either tamoxifen (TAM) or aromatase inhibitors is an important therapeutic strategy to treat or prevent estrogen receptor (ER) positive breast cancer. However, resistance to TAM is the major obstacle in endocrine therapy. This resistance occurs either de novo or is acquired after an initial beneficial response. The underlying mechanisms for TAM resistance are probably multifactorial and remain largely unknown. Considering that breast cancer is a very heterogeneous disease and patients respond differently to treatment, the molecular analysis of TAM's biological activity could provide the necessary framework to understand the complex effects of this drug in target cells. Moreover, this could explain, at least in part, the development of resistance and indicate an optimal therapeutic option. This review highlights the implications of TAM in breast cancer as well as the role of receptors/signal pathways recently suggested to be involved in the development of TAM resistance. G protein-coupled estrogen receptor, Androgen Receptor and Hedgehog signaling pathways are emerging as novel therapeutic targets and prognostic indicators for breast cancer, based on their ability to mediate estrogenic signaling in ERα-positive or -negative breast cancer.

Tamoxifen Treatment of Breast Cancer Cells: Impact on Hedgehog/GLI1 Signaling.

The selective estrogen receptor (ER) modulator tamoxifen (TAM) has become the standard therapy for the treatment of ER+ breast cancer patients. Despite the obvious benefits of TAM, a proportion of patients acquire resistance to treatment, and this is a significant clinical problem. Consequently, the identification of possible mechanisms involved in TAM-resistance should help the development of new therapeutic targets. In this study, we present in vitro data using a panel of different breast cancer cell lines and demonstrate the modulatory effect of TAM on cellular proliferation and expression of Hedgehog signaling components, including the terminal effector of the pathway, the transcription factor GLI1. A variable pattern of expression following TAM administration was observed, reflecting the distinctive properties of the ER+ and ER- cell lines analyzed. Remarkably, the TAM-induced increase in the proliferation of the ER+ ZR-75-1 and BT474 cells parallels a sustained upregulation of GLI1 expression and its translocation to the nucleus. These findings, implicating a TAM-GLI1 signaling cross-talk, could ultimately be exploited not only as a means for novel prognostication markers but also in efforts to effectively target breast cancer subtypes.

An updated h-index measures both the primary and total scientific output of a researcher.

The growing interest in scientometry stems from ethical concerns related to the proper evaluation of scientific contributions of an author working in a hard science. In the absence of a consensus, institutions may use arbitrary methods for evaluating scientists for employment and promotion. There are several indices in use that attempt to establish the most appropriate and suggestive position of any scientist in the field he/she works in. A scientist's Hirsch-index (h-index) quantifies their total effective published output, but h-index summarizes the total value of their published work without regard to their contribution to each publication. Consequently, articles where the author was a primary contributor carry the same weight as articles where the author played a minor role. Thus, we propose an updated h-index named Hirsch(p,t)-index that informs about both total scientific output and output where the author played a primary role. Our measure, h(p,t) = h(p),h(t), is composed of the h-index h(t) and the h-index calculated for articles where the author was a key contributor; i.e. first/shared first or senior or corresponding author. Thus, a h(p,t) = 5,10 would mean that the author has 5 articles as first, shared first, senior or corresponding author with at least 5 citations each, and 10 total articles with at least 10 citations each. This index can be applied in biomedical disciplines and in all areas where the first and last position on an article are the most important. Although other indexes, such as r- and w-indexes, were proposed for measuring the authors output based on the position of researchers within the published articles, our simpler strategy uses the already established algorithms for h-index calculation and may be more practical to implement.

Neighboring gene regulation by antisense long non-coding RNAs.

Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation--pretranscriptional, transcriptional and posttranscriptional--through DNA-RNA, RNA-RNA or protein-RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm.

The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling.

BACKGROUND: The crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. METHODS: siRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response. RESULTS: Although knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined. CONCLUSION: Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.

Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1.

Non-coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non-coding RNA located head-to-head with the gene encoding the Glioma-associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer-associated signaling pathways. The expression of this three-exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFß and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up-regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non-coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non-coding RNA-based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor.

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling.

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo.

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

Genetic variations and alternative splicing: the Glioma associated oncogene 1, GLI1.

Alternative splicing is a post-transcriptional regulatory process that is attaining stronger recognition as a modulator of gene expression. Alternative splicing occurs when the primary RNA transcript is differentially processed into more than one mature RNAs. This is the result of a variable definition/inclusion of the exons, the sequences that are excised from the primary RNA to form the mature RNAs. Consequently, RNA expression can generate a collection of differentially spliced RNAs, which may distinctly influence subsequent biological events, such as protein synthesis or other biomolecular interactions. Still the mechanisms that control exon definition and exon inclusion are not fully clarified. This mini-review highlights advances in this field as well as the impact of single nucleotide polymorphisms in affecting splicing decisions. The Glioma-associated oncogene 1, GLI1, is taken as an example in addressing the role of nucleotide substitutions for splicing regulation.

Novel mechanism of action on Hedgehog signaling by a suppressor of fused carboxy terminal variant.

The Suppressor of Fused (SUFU) protein plays an essential role in the Hedgehog (HH) signaling pathway, by regulation of the GLI transcription factors. Two major isoforms of human SUFU are known, a full-length (SUFU-FL) and a carboxy-terminal truncated (SUFU- ΔC) variant. Even though SUFU- ΔC is expressed at an equivalent level as SUFU-FL in certain tissues, the function of SUFU-ΔC and its impact on HH signal transduction is still unclear. In two cell lines from rhabdomyosarcoma, a tumor type associated with deregulated HH signaling, SUFU-ΔC mRNA was expressed at comparable levels as SUFU-FL mRNA, but at the protein level only low amounts of SUFU-ΔC were detectable. Heterologous expression provided support to the notion that the SUFU-ΔC protein is less stable compared to SUFU-FL. Despite this, biochemical analysis revealed that SUFU-ΔC could repress GLI2 and GLI1ΔN, but not GLI1FL, transcriptional activity to the same extent as SUFU-FL. Moreover, under conditions of activated HH signaling SUFU-ΔC was more effective than SUFU-FL in inhibiting GLI1ΔN. Importantly, co-expression with GLI1FL indicated that SUFU-ΔC but not SUFU-FL reduced the protein levels of GLI1FL. Additionally, confocal microscopy revealed a co-localization of GLI1FL with SUFU-ΔC but not SUFU-FL in aggregate structures. Moreover, specific siRNA mediated knock-down of SUFU-ΔC resulted in up-regulation of the protein levels of GLI1FL and the HH signaling target genes PTCH1 and HHIP. Our results are therefore suggesting the presence of novel regulatory controls in the HH signaling pathway, which are elicited by the distinct mechanism of action of the two alternative spliced SUFU proteins.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas.

BACKGROUND: Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. OBJECTIVES: To search for mutations and genomic instability in sporadic and inherited BCCs. METHODS: DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. RESULTS: Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g.2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g.2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. CONCLUSION: Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Basal cell carcinomas (BCCs) are the most frequent human cancer thatresults from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2.

Trans-splicing in Higher Eukaryotes: Implications for Cancer Development?

Trans-splicing, the possibility of exons from distinct pre-mRNAs to join together, is still a concept in gene expression that is generally regarded of limited significance. However, recent work has provided evidence that in human tumors trans-splicing events may precede chromosomal rearrangements. In fact, it has been suggested that the trans-spliced molecules could act as "guides" that facilitate the genomic translocation. This perspective highlights the development of the ideas of trans-splicing in higher eukaryotes during the last 25 years, from a bizarre phenomenon to a biological event that is attaining stronger recognition.

Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1.

BACKGROUND: Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. RESULTS: We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. CONCLUSIONS: Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.