Tsetse flies (Diptera: Glossinidae) house a taxonomically diverse microbiota that includes environmentally acquired bacteria, maternally transmitted symbiotic bacteria, and pathogenic African ...trypanosomes. Sodalis glossinidius, which is a facultative symbiont that resides intra and extracellularly within multiple tsetse tissues, has been implicated as a mediator of trypanosome infection establishment in the fly's gut. Tsetse's gut-associated population of Sodalis are subjected to marked temperature fluctuations each time their ectothermic fly host imbibes vertebrate blood. The molecular mechanisms that Sodalis employs to deal with this heat stress are unknown. In this study, we examined the thermal tolerance and heat shock response of Sodalis. When grown on BHI agar plates, the bacterium exhibited the most prolific growth at 25oC, and did not grow at temperatures above 30oC. Growth on BHI agar plates at 31°C was dependent on either the addition of blood to the agar or reduction in oxygen levels. Sodalis was viable in liquid cultures for 24 hours at 30oC, but began to die upon further exposure. The rate of death increased with increased temperature. Similarly, Sodalis was able to survive for 48 hours within tsetse flies housed at 30oC, while a higher temperature (37oC) was lethal. Sodalis' genome contains homologues of the heat shock chaperone protein-encoding genes dnaK, dnaJ, and grpE, and their expression was up-regulated in thermally stressed Sodalis, both in vitro and in vivo within tsetse fly midguts. Arrested growth of E. coli dnaK, dnaJ, or grpE mutants under thermal stress was reversed when the cells were transformed with a low copy plasmid that encoded the Sodalis homologues of these genes. The information contained in this study provides insight into how arthropod vector enteric commensals, many of which mediate their host's ability to transmit pathogens, mitigate heat shock associated with the ingestion of a blood meal.
FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present ...a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis.
For complete details on the use and execution of this protocol, please refer to Antony et al. (2022).1
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•Combination of FISH and flow cytometry to quantify nascent and mature ribosomal RNA•Quantification of rRNA in individual cells and in cell cycle phases•Probe sequences designed and tested for human and mouse cells
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FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis.
PHF6 is an X-chromosome gene showing recurrent loss-of-function mutations in acute and chronic myeloid leukemias and T-lymphoblastic leukemia, indicating that it acts as a tumor suppressor in both ...myeloid and lymphoid hematopoietic lineages. PHF6 protein is localized to the nucleolus, the site of ribosome biogenesis, where it is reported to regulate rDNA transcription. It is also localized to the nucleoplasm, where it binds chromatin and may regulate gene transcription. However, these mechanisms are incompletely established, no animal model of PHF6 loss has been reported, and there is limited insight into the precise role of PHF6 in hematopoiesis, both mechanistically and as a leukemia suppressor.
To study the in vivo role of Phf6, we generated mice with hematopoietic knockout of Phf6 using the Vav-Cre recombinase system, achieving a >95% deletion efficiency. In comparison with Vav-Cre mice (WT), mice with Vav-Cre;Phf6-flox genotype (Phf6 KO) showed, at 8-12 weeks of age, a 1.25-fold expansion of the LSK (Lin-Sca1+Kit+) compartment in the bone marrow (see figure), accompanied with a similar increase in the common myeloid progenitor CMP compartment (Lin-Kit+Sca1-CD34+FcRIII-). Within the LSK compartment, there was a 2-fold and 1.7-fold expansion of the myeloid-biased multipotent progenitor compartments MPP2 (LSK,Flk2-CD150+CD48+) and MPP3 (LSK,Flk2-CD150-CD48+) respectively. The lymphoid-biased MPP4 compartment was not changed, nor was the common lymphoid progenitor CLP compartment (not shown). Conversely, the number of stringently defined HSCs (LSK,Flk2-CD150+CD48-CD34-) was reduced by 40%. This suggests depletion of HSCs through loss of dormancy, accompanied by myeloid skewing. At 8-12 weeks of age, there was no change in overall bone marrow cellularity or spleen size/cellularity, though flowcytometric analysis of spleen showed identical reduction of HSC and expansion of MPP2 compartments in Phf6 KO. As of 40 weeks of age, Phf6 KO mice did not show any gross peripheral blood count abnormalities.
We also used CRISPR/Cas9 to generate PHF6 knockout clones from the THP-1 human AML cell line. RNA-Seq and quantitative proteomics in knockout cells showed downregulation of mature myeloid genes and increased expression of hematopoietic progenitor gene sets, including increased expression of cell surface receptor KIT. KO cells showed increased proliferation when cultured with KIT ligand. Using IP-mass spectrometry in WT and KO clones, we identified ribosomal proteins RPL12 and RPLP0 as the most abundant and specific binding partners of PHF6.
In summary, young Phf6 knockout mice show HSC depletion and expansion of myeloid-skewed progenitors without overt peripheral blood abnormalities. Further work is in progress to characterize HSC dormancy and competitiveness, progression of Phf6 KO phenotype with age, and mechanisms of gene regulation by Phf6 through binding of ribosomal proteins.
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No relevant conflicts of interest to declare.
Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA ...transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.
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•Multiple cell-type-specific TFs bind canonical motifs on rDNA•The hematopoietic TF CEBPA binds to active rDNA alleles at a conserved site•CEBPA promotes RNA Pol I occupancy and rRNA transcription in myeloid progenitors•We present “47S-FISH-Flow,” a sensitive assay to quantify nascent rRNA
Antony et al. mapped existing ChIP-seq datasets to human and mouse rDNA and identified binding profiles of cell-type-specific transcription factors (TFs). The myeloid TF CEBPA bound rDNA at a conserved motif, and its degradation reduced rDNA occupancy of the RNA Pol I-RRN3 complex and nascent 47S rRNA transcription.
Alkyl radicals are prominent in combustion chemistry as they are formed by hydrocarbon decomposition or from a radical attack on hydrocarbons. Accurate determinations of the thermochemistry and ...kinetics of their unimolecular isomerization and decomposition reactions and related addition reactions of alkenes are therefore important in simulating the combustion chemistry of virtually all hydrocarbon fuels. In this work, a comprehensive potential energy surface (PES) for Ḣ-atom addition to and abstraction from 1- and 2-pentene, and the subsequent C–C and C–H β-scission reactions, and H-atom transfer reactions has been considered. Thermochemical values for the species on the Ċ5H11 PES were calculated as a function of temperature (298–2000 K), with enthalpies of formation determined using a network of isodesmic reactions. High-pressure limiting and pressure-dependent rate constants were calculated using the Rice-Ramsperger-Kassel-Marcus theory coupled with a one-dimensional master equation. As a validation of our theoretical results, hydrogen atomic resonance absorption spectrometry experiments were performed on the Ḣ-atom addition and abstraction reactions of 1- and 2-pentene. By incorporating our calculations into a detailed chemical kinetic model (AramcoMech 3.0), excellent agreement with these experiments is observed. The theoretical results are further validated via a comprehensive series of simulations of literature data. Our a priori model is found to reproduce important absolute species concentrations and product ratios reported therein.
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