Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome.

Neurons express various combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here, we use epitope-tagged endogenous NR subunits, expansion light-sheet microscopy, and electron microscopy (EM) connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion-sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type-specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determine patterns of synaptic inputs. In support of this model, we identify a transmembrane protein selectively associated with a subset of spatially restricted synapses and demonstrate its requirement for synapse formation through genetic analysis. We propose that mechanisms that regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome.

Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here we use epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, we identify a transmembrane protein associated selectively with a subset of spatially restricted synapses and demonstrate through genetic analysis its requirement for synapse formation. We propose that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

Analyzing Iron and Oxygen-Regulated Protein Complex Formation Using Proteomic Mass Spectrometry.

Multicellular organisms have evolved elaborate strategies to sense and adapt to changes in intracellular oxygen. The canonical cellular pathway responsible for oxygen sensing consists of the von Hippel-Lindau (pVHL) tumor suppressor protein, prolyl hydroxylases (PHD), and hypoxia-inducible factors (HIFs), which together regulate expression of downstream genes involved in oxygen homeostasis. In recent years, it has become increasingly clear that oxygen regulatory mechanisms are intertwined with cellular iron-sensing pathways. Key members of these networks such as prolyl-hydroxylases, E3 ubiquitin ligase adaptor protein FBXL5, iron regulatory proteins (IRPs), and Fe-S cluster proteins require both iron and oxygen for their optimal function and/or are tightly regulated by intracellular concentrations of these molecules. Monitoring how protein interactomes are remodeled as a function of intracellular oxygen and iron levels gives insights into the nature and dynamics of these pathways. We have recently described an oxygen-sensitive interaction between FBXL5 and the cytoplasmic Fe-S cluster targeting complex (CIA targeting complex) with implications in the FBXL5-dependent regulation of IRPs. Based on this work, we present a protocol describing the induction and maintenance of hypoxia in mammalian cell cultures and a mass-spectrometry-based proteomics approach aimed at interrogating changes in interactome of key proteins as a function of intracellular oxygen and iron levels. These methods are widely applicable to understanding the dynamics of iron and oxygen signaling.

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing.

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns.

The high complexity prevalent in biological samples requires chromatographic separations with high sensitivity and resolution to be effectively analyzed. Here we introduce a robust, reproducible and inexpensive protocol for preparation of a nano-flow reversed phase high performance liquid chromatography (RP-HPLC) columns for on-line separation of analytical peptides before introduction into and detection by a mass-spectrometer in traditional bottom-up proteomics workflows. Depending on the goal of the experiment and the chemical properties of the analytes being separated, optimal column parameters may differ in their internal or outer diameters, length, particle size, pore size, chemistry of stationary phase particles, and the presence or absence of an integrated electrospray emitter at the tip. An in-house column packing system not only enables the rapid fabrication of columns with the desired properties but also dramatically reduces the cost of the process. The optimized protocol for packing a C18 AQ (aqueous) fused silica column discussed here is compatible with a wide range of liquid chromatographic instruments for achieving effective separation of analytes.

Author Correction: Stabilization of SIRT7 deacetylase by viral oncoprotein HBx leads to inhibition of growth restrictive RPS7 gene and facilitates cellular transformation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation.

Stabilization of SIRT7 deacetylase by viral oncoprotein HBx leads to inhibition of growth restrictive RPS7 gene and facilitates cellular transformation.

Sirtuin-7 (SIRT7) deacetylase exhibits a high selectivity for acetylated H3K18 and has been implicated in the maintenance of malignant phenotype. However, it remains unclear if SIRT7 and H3K18ac play a role in the tumorigenic program driven by oncogenic viruses. We show that ectopically expressed HBx oncoprotein of hepatitis B virus promoted intracellular stability of SIRT7 by salvaging it from ubiquitin-mediated proteasomal degradation. HBx-dependent accumulation of SIRT7 favored H3K18 deacetylation and down-regulated the small ribosomal protein gene, RPS7, involved in cell death and DNA damage response. HBx facilitated the recruitment of SIRT7 to RPS7 promoter thus impeding H3K18ac occupancy and hindering RPS7 transcription. The antagonistic relationship between SIRT7 and RPS7 was also observed in the HBx transgenic mice, where elevated levels of SIRT7 protein were coincident with low levels of H3K18ac and RPS7. Strikingly, inhibition of cellular deubiquitinase activity restored RPS7 gene transcription. Further, depletion of endogenous SIRT7 led to decreased cell viability and transformation. The biological relevance of RPS7 suppression by HBx-SIRT7 axis was evident from ectopic expression of RPS7 which attenuated clonogenicity of cells. Thus, our findings suggest that SIRT7 is a critical regulator of HBx-driven oncogenic program, through its antagonistic impact on growth restrictive ribosomal protein RPS7.

The "Macro" World of microRNAs in Hepatocellular Carcinoma.

Hepatotropic viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) are the major etiological agents associated with development of hepatocellular carcinoma (HCC). Progression of HCC is a multistep process that requires sequential or parallel deregulation of oncogenic and tumor suppressive pathways leading to chromosomal instability and neoplastic phenotype. In the recent years, microRNAs (miRNAs) have carved their own niche alongside oncogenes and tumor suppressors, owing to their innate ability to receive and relay multiple signals. Not surprisingly, miRNAs are fast emerging as central player in myriads of malignancies including HCC. miRNAs are reported to participate in initiation and progression of HCC, and have also been clinically correlated with risk assessment, disease grade, aggressiveness, and prognosis. Despite extensive data available on the role of miRNAs in HCC, there is a pressing need to integrate and evaluate these datasets to find its correlation, if any, with causal agents in order to devise novel interventional modalities. Through this review, we attempt to bridge the gap by consolidating the current knowledge and concepts in the field of HCC-related miRNAs with special emphasis on HBV and HCV. Further, we assess the potential of common as well as unique signatures that may be useful in developing novel biomarkers and therapeutics.

HBx protein of hepatitis B virus promotes reinitiation of DNA replication by regulating expression and intracellular stability of replication licensing factor CDC6.

Prevention of re-replication via negative regulation of replication initiator proteins, such as CDC6, is key to maintenance of genomic integrity, whereas their up-regulation is generally associated with perturbation in cell cycle, genomic instability, and potentially, tumorigenesis. The HBx oncoprotein of hepatitis B virus is well known to deregulate cell cycle and has been intricately linked to development of hepatocellular carcinoma. Despite a clear understanding of the proliferative effects of HBx on cell cycle, a mechanistic link between HBx-mediated hepatocarcinogenesis and host cell DNA replication remains poorly perused. Here we show that HBx overexpression in both the cellular as well as the transgenic environment resulted in the accumulation of CDC6 through transcriptional and post-translational up-regulation. The HBx-mediated increase in CDK2 activity altered the E2F1-Rb (retinoblastoma) balance, which favored CDC6 gene expression by E2F1. Besides, HBx impaired the APC(Cdh1)-dependent protein degradation pathway and conferred intracellular stability to CDC6 protein. Increase in CDC6 levels correlated with increase in CDC6 occupancy on the ß-globin origin of replication, suggesting increment in origin licensing and re-replication. In conclusion, our findings strongly suggest a novel role for CDC6 in abetting the oncogenic sabotage carried out by HBx and support the paradigm that pre-replicative complex proteins have a role in oncogenic transformation.