Delivery of Targeted Co(III)-DNA Inhibitors of Gli Proteins to Disrupt Hedgehog Signaling.

The Hedgehog (Hh) signaling pathway is integral for embryonic development and normal cell maintenance. However, aberrant expression of the Hh pathway is recognized as the oncogenic driving force for basal cell carcinoma (BCC). Current chemotherapeutic treatments that inhibit Hh signaling allow treatment of only locally advanced and metastatic BCCs via inhibition of the transmembrane protein, smoothened. It is further recognized that downstream mutations often lead to chemoresistant tumor recurrence. The Gli proteins are the ultimate regulators of Hh signaling and belong to a family of Cys2His2 zinc finger transcription factors (ZnFTFs) that we have shown can be irreversibly inhibited by a series of cobalt(III) Schiff base-DNA (CoSB-DNA) conjugates. However, a significant challenge is the delivery of CoSB-DNA complexes in mammalian tissues. Herein, we report a polyethyleneimine-functionalized graphene oxide nanoconjugate (GOPEI) that delivers CoGli, a CoSB-DNA complex that targets Gli specifically. We describe the characterization of the surface functionalization of GOPEI and accumulation in ASZ murine BCC cells via confocal microscopy and inductively coupled plasma-mass spectrometry (ICP-MS). Lysosomal escape of CoGli is further confirmed by confocal microscopy. We report the successful targeting of Gli by CoGli and a 17-fold improvement in potency over small-molecule Gli inhibitor GANT-61 in inhibiting Hh-driven migration of ASZ murine BCC cells. This study provides a promising starting point for further investigating CoGli inhibitors of Hh signaling in developed mammalian tissues.

Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels.

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. Here, we show that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, we provide evidence that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.

Functional Disruption of Gli1-DNA Recognition via a Cobalt(III) Complex.

The aberrant activation of the Gli family of zinc finger transcription factors (ZFTFs) is associated with several types of human cancer, including medulloblastoma and basal cell carcinoma. We have reported the use of cobalt(III) Schiff-base complexes (Co(III)-sb) as potent inhibitors of ZFTFs in vivo. These complexes inhibit transcription by displacing the zinc finger domain's structural Zn(II) ion, destabilizing the alpha helix necessary for DNA recognition. Here, we describe the use of Co(III)-sb complexes for the selective inhibition of Gli1. Spectroscopic and computational studies of the Gli1 DNA binding domain found that Co(III)-sb displaced Zn(II) through direct coordination with the His residues of the Cys2 His2 Zn(II) binding site. As a result, there is a dose-dependent degradation of the alpha-helix content in the DNA binding domain of Gli1 and corresponding inhibition of consensus sequence recognition. We conclude that this strategy is well suited for the development of new and potent inhibitors of Gli1.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases.

The conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila, the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them-SMURF and NEDD4-are known to regulate its levels. We demonstrate that mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, we show that the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases.

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning.

Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the -1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.

Specific inhibition of the transcription factor Ci by a cobalt(III) Schiff base-DNA conjugate.

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well-known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci's consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anticancer therapeutics.

A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome.

The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome.

Regulation of Ci and Su(fu) nuclear import in Drosophila.

The Hedgehog (Hh) signal transduction pathway plays a central role in the development of invertebrates and vertebrates. While much is known about the pathway, the role of Suppressor of fused (Su(fu)), a component of the pathway's signaling complex has remained enigmatic. Previous studies have linked Su(fu) to the cytoplasmic sequestration of the zinc finger transcription factor, Cubitus interruptus (Ci), while other studies suggest a role in modulating target gene expression. In examining the cell biology of the pathway, we have found that like its vertebrate homologue, Drosophila Su(fu) enters the nucleus. Furthermore, we find that the nuclear import of Su(fu) occurs in concert with that of Ci in response to Hh signaling. Here, we examine the mechanism by which Su(fu) regulates Ci import by investigating the importance of the Ci nuclear localization signal (NLS) and the effect of adding an additional NLS. Finally, we demonstrate that Ci can bring Su(fu) with it to a multimerized Ci DNA binding site. These results provide a basis for understanding the dual roles played by Su(fu) in the regulation of Ci.

Regulation of the Drosophila transcription factor, Cubitus interruptus, by two conserved domains.

Hedgehog signaling is required for the development of many organisms, including Drosophila. In flies, Hh patterns the embryonic epidermis and larval imaginal discs by regulating the transcription factor, Cubitus interruptus (Ci). To date, three levels of regulation have been identified: proteolytic processing into a repressor, nuclear import, and activation. In this report, we characterize the function of two Ci domains that are conserved in the vertebrate homologues, GLI1, GLI2, and GLI3. One domain includes the first two of five C(2)-H(2) zinc-fingers. While conserved in all members of the GLI/Ci family, the first two fingers do not appear to make significant contacts with the DNA target sequence. Ci protein lacking this region is still able to interact with the cytoplasmic complex and activate transcription in embryos and wing imaginal discs, but it is no longer processed into the repressor form. The second domain, termed NR for "N-terminal Regulatory", binds Suppressor of Fused. Deletion of this region has little effect on embryonic patterning, but compromises cytoplasmic retention of Ci. Analysis of the amino acid sequence of this domain identifies 11 perfectly conserved serines and one tyrosine. We propose that this region may be modified, possibly by phosphorylation, to regulate Ci nuclear import.

A conserved processing mechanism regulates the activity of transcription factors Cubitus interruptus and NF-kappaB.

The proteasome degrades some proteins, such as transcription factors Cubitus interruptus (Ci) and NF-kappaB, to generate biologically active protein fragments. Here we have identified and characterized the signals in the substrate proteins that cause this processing. The minimum signal consists of a simple sequence preceding a tightly folded domain in the direction of proteasome movement. The strength of the processing signal depends primarily on the complexity of the simple sequence rather than on amino acid identity, the resistance of the folded domain to unraveling by the proteasome and the spacing between the simple sequence and folded domain. We show that two unrelated transcription factors, Ci and NF-kappaB, use this mechanism to undergo partial degradation by the proteasome in vivo. These findings suggest that the mechanism is conserved evolutionarily and that processing signals may be widespread in regulatory proteins.

Ci proteolysis: regulation by a constellation of phosphorylation sites.

Proteolytic processing of Ci requires phosphorylation by the kinases Sgg/GSK3 and CK1. The newly identified phosphorylation sites form clusters with previously described PKA sites in which phosphorylation by PKA acts to prime subsequent phosphorylation by Sgg/GSK3 and CK1.