Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms.

Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc's) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc's, and a TOP1 mutation (T718A) that stabilizes TOP1cc's. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.

GABAergic Neuron-Specific Loss of Ube3a Causes Angelman Syndrome-Like EEG Abnormalities and Enhances Seizure Susceptibility.

Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)-all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

EGFR and FGFR pathways have distinct roles in Drosophila mushroom body development and ethanol-induced behavior.

Epidermal Growth Factor Receptor (EGFR) signaling has a conserved role in ethanol-induced behavior in flies and mice, affecting ethanol-induced sedation in both species. However it is not known what other effects EGFR signaling may have on ethanol-induced behavior, or what roles other Receptor Tyrosine Kinase (RTK) pathways may play in ethanol induced behaviors. We examined the effects of both the EGFR and Fibroblast Growth Factor Receptor (FGFR) RTK signaling pathways on ethanol-induced enhancement of locomotion, a behavior distinct from sedation that may be associated with the rewarding effects of ethanol. We find that both EGFR and FGFR genes influence ethanol-induced locomotion, though their effects are opposite - EGFR signaling suppresses this behavior, while FGFR signaling promotes it. EGFR signaling affects development of the Drosophila mushroom bodies in conjunction with the JNK MAP kinase basket (bsk), and with the Ste20 kinase tao, and we hypothesize that the EGFR pathway affects ethanol-induced locomotion through its effects on neuronal development. We find, however, that FGFR signaling most likely affects ethanol-induced behavior through a different mechanism, possibly through acute action in adult neurons.

Topoisomerases facilitate transcription of long genes linked to autism.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord.

Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E) hydrolyze extracellular AMP to adenosine in dorsal root ganglia (DRG) neurons and in the dorsal spinal cord. Previously, we found that adenosine production was reduced, but not eliminated, in Pap⁻/⁻/Nt5e⁻/⁻ double knock-out (dKO) mice, suggesting that a third AMP ectonucleotidase was present in these tissues. Here, we found that tissue-nonspecific alkaline phosphatase (TNAP, encoded by the Alpl gene) is expressed and functional in DRG neurons and spinal neurons. Using a cell-based assay, we found that TNAP rapidly hydrolyzed extracellular AMP and activated adenosine receptors. This activity was eliminated by MLS-0038949, a selective pharmacological inhibitor of TNAP. In addition, MLS-0038949 eliminated AMP hydrolysis in DRG and spinal lamina II of dKO mice. Using fast-scan-cyclic voltammetry, we found that adenosine was rapidly produced from AMP in spinal cord slices from dKO mice, but virtually no adenosine was produced in spinal cord slices from dKO mice treated with MLS-0038949. Last, we found that AMP inhibited excitatory neurotransmission via adenosine A1 receptor activation in spinal cord slices from wild-type, Pap⁻/⁻, Nt5e⁻/⁻, and dKO mice, but failed to inhibit neurotransmission in slices from dKO mice treated with MLS-0038949. These data suggest that triple elimination of TNAP, PAP, and NT5E is required to block AMP hydrolysis to adenosine in DRG neurons and dorsal spinal cord. Moreover, our data reveal that TNAP, PAP, and NT5E are the main AMP ectonucleotidases in primary somatosensory neurons and regulate physiology by metabolizing extracellular purine nucleotides.

Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons.

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.

Selection of appropriate oviposition sites is essential for progeny survival and fitness in generalist insect species, such as Drosophila melanogaster, yet little is known about the mechanisms regulating how environmental conditions and innate adult preferences are evaluated and balanced to yield the final substrate choice for egg-deposition. Female D. melanogaster are attracted to food containing acetic acid (AA) as an oviposition substrate. However, our observations reveal that this egg-laying preference is a complex process, as it directly opposes an otherwise strong, default behavior of positional avoidance for the same food. We show that 2 distinct sensory modalities detect AA. Attraction to AA-containing food for the purpose of egg-laying relies on the gustatory system, while positional repulsion depends primarily on the olfactory system. Similarly, distinct central brain regions are involved in AA attraction and repulsion. Given this unique situation, in which a single environmental stimulus yields 2 opposing behavioral outputs, we propose that the interaction of egg-laying attraction and positional aversion for AA provides a powerful model for studying how organisms balance competing behavioral drives and integrate signals involved in choice-like processes.

Analysis of a polycomb group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.

Polycomb group (PcG) genes propagate patterns of transcriptional repression throughout development. The products of several such genes are part of Polycomb repressive complex 1 (PRC1), which inhibits chromatin remodeling and transcription in vitro. Genetic and biochemical studies suggest the product of the Posterior sex combs (Psc) gene plays a central role in both PcG-mediated gene repression in vivo and PRC1 activity in vitro. To dissect the relationship between the in vivo and in vitro activities of Psc, we identified the lesions associated with 11 genetically characterized Psc mutations and asked how the corresponding mutant proteins affect Psc activity on nucleosomal templates in vitro. Analysis of both single-mutant Psc proteins and recombinant complexes containing mutant protein revealed that Psc encodes at least two functions, complex formation and the inhibition of remodeling and transcription, which require different regions of the protein. There is an excellent correlation between the in vivo phenotypes of mutant Psc alleles and the structure and in vitro activities of the corresponding proteins, suggesting that the in vitro activities of PRC1 reflect essential functions of Psc in vivo.

Division of labor in polycomb group repression.

The epigenetic maintenance of gene expression patterns is essential for developing and maintaining the diverse types of cell that cooperate to form the larger organism. Recent data suggest that proteins of the Polycomb group (PcG) use a combination of posttranslational modifications and structural changes to the underlying chromatin structure to maintain silenced epigenetic states. We are now beginning to understand the mechanisms by which the PcG proteins are able to silence genes and to maintain this silencing over many cell divisions.

Propagation of silencing; recruitment and repression of naive chromatin in trans by polycomb repressed chromatin.

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.

Regulation of Polycomb group complexes by the sequence-specific DNA binding proteins Zeste and GAGA.

Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. We have used purified proteins and complexes reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1.

Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro.

Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. We have established an in vitro system in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC) containing only PcG proteins are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. We find that assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction.