Proteasome gene expression is controlled by coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6's role in hematopoietic differentiation and leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources). METHODS: INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo. RESULTS: The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo. CONCLUSIONS: Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Decidual-tissue-resident memory T cells protect against nonprimary human cytomegalovirus infection at the maternal-fetal interface.

Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employ an ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity against CMV, recapitulating nonprimary vs. primary infection at the authentic maternofetal transmission site. We show that decidual tissues of women with preconception immunity against CMV exhibit intrinsic resistance to HCMV, mounting a rapid activation of tissue-resident memory CD8+ and CD4+ T cells upon HCMV reinfection. We further reveal the role of HCMV-specific decidual-tissue-resident CD8+ T cells in local protection against nonprimary HCMV infection. The findings could inform the development of a vaccine against cCMV and provide insights for further studies of the integrity of immune defense against HCMV and other pathogens at the human maternal-fetal interface.

Proteasome gene expression is controlled by the coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo , we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 d after denervation) it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PAL NRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PAL NRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g. type-2 diabetes, cancer).

Genome-scale reconstruction of Paenarthrobacter aurescens TC1 metabolic model towards the study of atrazine bioremediation.

Atrazine is an herbicide and a pollutant of great environmental concern that is naturally biodegraded by microbial communities. Paenarthrobacter aurescens TC1 is one of the most studied degraders of this herbicide. Here, we developed a genome scale metabolic model for P. aurescens TC1, iRZ1179, to study the atrazine degradation process at organism level. Constraint based flux balance analysis and time dependent simulations were used to explore the organism's phenotypic landscape. Simulations aimed at designing media optimized for supporting growth and enhancing degradation, by passing the need in strain design via genetic modifications. Growth and degradation simulations were carried with more than 100 compounds consumed by P. aurescens TC1. In vitro validation confirmed the predicted classification of different compounds as efficient, moderate or poor stimulators of growth. Simulations successfully captured previous reports on the use of glucose and phosphate as bio-stimulators of atrazine degradation, supported by in vitro validation. Model predictions can go beyond supplementing the medium with a single compound and can predict the growth outcomes for higher complexity combinations. Hence, the analysis demonstrates that the exhaustive power of the genome scale metabolic reconstruction allows capturing complexities that are beyond common biochemical expertise and knowledge and further support the importance of computational platforms for the educated design of complex media. The model presented here can potentially serve as a predictive tool towards achieving optimal biodegradation efficiencies and for the development of ecologically friendly solutions for pollutant degradation.

Modeling trophic dependencies and exchanges among insects' bacterial symbionts in a host-simulated environment.

BACKGROUND: Individual organisms are linked to their communities and ecosystems via metabolic activities. Metabolic exchanges and co-dependencies have long been suggested to have a pivotal role in determining community structure. In phloem-feeding insects such metabolic interactions with bacteria enable complementation of their deprived nutrition. The phloem-feeding whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) harbors an obligatory symbiotic bacterium, as well as varying combinations of facultative symbionts. This well-defined bacterial community in B. tabaci serves here as a case study for a comprehensive and systematic survey of metabolic interactions within the bacterial community and their associations with documented occurrences of bacterial combinations. We first reconstructed the metabolic networks of five common B. tabaci symbionts genera (Portiera, Rickettsia, Hamiltonella, Cardinium and Wolbachia), and then used network analysis approaches to predict: (1) species-specific metabolic capacities in a simulated bacteriocyte-like environment; (2) metabolic capacities of the corresponding species' combinations, and (3) dependencies of each species on different media components. RESULTS: The predictions for metabolic capacities of the symbionts in the host environment were in general agreement with previously reported genome analyses, each focused on the single-species level. The analysis suggests several previously un-reported routes for complementary interactions and estimated the dependency of each symbiont in specific host metabolites. No clear association was detected between metabolic co-dependencies and co-occurrence patterns. CONCLUSIONS: The analysis generated predictions for testable hypotheses of metabolic exchanges and co-dependencies in bacterial communities and by crossing them with co-occurrence profiles, contextualized interaction patterns into a wider ecological perspective.

Host-Parasite-Bacteria Triangle: The Microbiome of the Parasitic Weed Phelipanche aegyptiaca and Tomato-Solanum lycopersicum (Mill.) as a Host.

Broomrapes (Phelipanche/Orobanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops, establishing direct connections with the host vascular system. This connection allows for the exchange of various substances and a possible exchange of endophytic microorganisms that inhabit the internal tissues of both plants. To shed some light on bacterial interactions occurring between the parasitic Phelipanche aegyptiaca and its host tomato, we characterized the endophytic composition in the parasite during the parasitization process and ascertained if these changes were accompanied by changes to endophytes in the host root. Endophyte communities of the parasitic weed were significantly different from that of the non-parasitized tomato root but no significant differences were observed between the parasite and its host after parasitization, suggesting the occurrence of bacterial exchange between these two plants. Moreover, the P. aegyptiaca endophytic community composition showed a clear shift from gram negative to gram-positive bacteria at different developmental stages of the parasite life cycle. To examine possible functions of the endophytic bacteria in both the host and the parasite plants, a number of unique bacterial candidates were isolated and characterized. Results showed that a Pseudomonas strain PhelS10, originating from the tomato roots, suppressed approximately 80% of P. aegyptiaca seed germination and significantly reduced P. aegyptiaca parasitism. The information gleaned in the present study regarding the endophytic microbial communities in this unique ecological system of two plants connected by their vascular system, highlights the potential of exploiting alternative environmentally friendly approaches for parasitic weed control.

Multidimensional patterns of metabolic response in abiotic stress-induced growth of Arabidopsis thaliana.

KEY MESSAGE: Contextualization of specific transcriptional responses of Arabidopsis within the stress-tissue-time perspective provides a simplified representation of the cellular transcriptional response pathways to abiotic stress, while reducing the dimensions in gene-oriented response description. Crops resistant to abiotic stresses are a long-term goal of many research programs, thus understanding the progression of stress responses is of great interest. We reanalyzed the AtGenExpress transcription dataset to go beyond gene-level characterization, and to contextualize the discrete information into (1) a process-level signature of stress-specific, time-specific, and tissue-specific responses and (2) identify patterns of response progression across a time axis. To gain a functional perspective, ∼1000 pathways associated with the differentially-expressed genes were characterized across all experiments. We find that the global response of pathways to stress is multi-dimensional and does not obviously cluster according to stress, time or tissue. The early response to abiotic stress typically involves induction of genes involved in transcription, hormone synthesis and signaling modules; a later response typically involves metabolism of amino acids and secondary metabolites. By linking specific primary and secondary response pathways, we outline possible stress-associated routes of response progression. The contextualization of specific processes within stress-tissue-time perspective provides a simplified representation of cellular response while reducing the dimensions in gene-oriented response description. Such simplified representation allows finding stress-specific markers based on process-combinations pointing whether a stress-specific response was invoked as well as provide a reference point for the conductance of comparative inter-plant study of stress response, bypassing the need in detailed orthologous mapping.

Draft Genome Sequence of a Dyella-Like Bacterium from the Planthopper Hyalesthes obsoletus.

We report here the draft genome sequence of a Dyella-like bacterium (DLB) isolated from Hyalesthes obsoletus, the insect vector of the uncultivable mollicute bacterium "Candidatus Phytoplasma." This isolate inhibits Spiroplasma melliferum, a cultivable mollicute. The draft genome of DLB consists of 4,196,214 bp, with a 68.6% G+C content, and 3,757 genes were predicted.

Variations in the identity and complexity of endosymbiont combinations in whitefly hosts.

The target of natural selection is suggested to be the holobiont - the organism together with its associated symbiotic microorganisms. The well-defined endosymbiotic communities of insects make them a useful model for exploring the role of symbiotic interactions in shaping the functional repertoire of plants and animals. Here, we studied the variations in the symbiotic communities of the sweet potato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) by compiling a dataset of over 2000 individuals derived from several independent screenings. The secondary endosymbionts harbored by each individual were clustered into entities termed Facultative Endosymbiont Combinations (FECs), each representing a natural assemblage of co-occurring bacterial genera. The association of FECs with whitefly individuals stratified the otherwise homogeneous population into holobiont units. We both identified bacterial assemblages that are specific to whitefly groups sharing unique genetic backgrounds, and characterized the FEC variations within these groups. The analysis revealed that FEC complexity is positively correlated with both distance from the equator and specificity of the genetic clade of the host insect. These findings highlight the importance of symbiotic combinations in shaping the distribution patterns of B. tabaci and possibly other insect species.

Neocentromeres form efficiently at multiple possible loci in Candida albicans.

Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Delta::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Delta::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: "proximal neoCEN" and "distal neoCEN" strains. Neocentromeres in the distal neoCEN strains formed at loci about 200-450 kb from cen5Delta::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-A(Cse4p) chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Delta::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.