Biology subjects

Hua, B.

Publications and source records attributed to Hua, B..

10 recordsLinked to original sources

bioRxiv2024

McsB regulates CtsR thermosensing through periphery arginine phosphorylation

When cells sense an elevated temperature in the environment, the bacterial master transcription repressor CtsR becomes phosphorylated and inactivated by the arginine kinase McsB to initiate the expression of heat-shock genes. Here, we utilize a fluorescence intensity shift assay (FISA) based on the protein-induced fluorescence enhancement (PIFE) effect to monitor the DNA-CtsR-McsB interactions in real time. Our single-molecule analysis reveals that CtsR binds rapidly and stably to the cognate DNA, and that McsB is able to transiently interact with CtsR in situ of the target DNA. We determine the binding kinetics between McsB and the DNA-bound CtsR by single-molecule real-time binding assays, with kon and koff of 0.75 M-1 s-1 and 0.34 s-1, respectively. This interaction with McsB does not remove CtsR from the DNA, but lowers the temperature threshold for CtsR dissociation and alters its thermosensing behavior. Mass spectrometry, mutational analysis and structural simulation results together suggest that the phosphorylation of several periphery arginine residues on CtsR, which reduces the binding energy of the CtsR-DNA interaction, underlies a plausible molecular mechanism for this effect. Taken together, these results provide insights into how McsB regulates the CtsR-DNA complex and highlight the functional importance of CtsR periphery arginine residues in the bacterial heat-shock response.

biophysics↗

bioRxiv2022

Genomewide identification of subtelomeric silencing factors in budding yeast

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and aging. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study subtelomeric gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a genome-wide, comprehensive screen for subtelomeric-silencing factors. The approach was replicable and allowed detection of expression changes caused by previously described silencing factors. We also identified new molecular players affecting this process, most of which are related to functions underlying chromatin conformation. This was the case of LGE1, a novel silencing factor herein reported, associated with histone ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

genetics↗

bioRxiv2021

A lipid-mTORC1 nutrient-sensing pathway regulates animal development by peroxisome-derived hormones

Animals have developed many signaling mechanisms that alter cellular and developmental programs in response to changes in nutrients and their derived metabolites, many of which remain to be understood. We recently uncovered that glucosylceramides, a core sphingolipid, act as a critical nutrient signal for overall amino-acid level to promote development by activating the intestinal mTORC1 pathway. However, how the intestinal GlcCer-mTORC1 activity regulates development throughout the whole body is unknown. Through a large-scale genetic screen, we found that the peroxisomes are critical for antagonizing the GlcCer-mTORC1-mediated nutrient signal. Mechanistically, deficiency of glucosylceramide, inactivation of the downstream mTORC1 activity, or prolonged starvation relocated peroxisomes closer to the intestinal apical region to release peroxisomal-beta-oxidation derived hormones that targeting chemosensory neurons to arrest the animal development. Our data illustrated a new gut-brain axis for orchestrating nutrient-sensing dependent development in Caenorhabditis elegans, which may also explain why glucosylceramide and peroxisome become essential in metazoans.

cell biology↗

bioRxiv2021

Computational analysis of GAL pathway pinpoints mechanisms underlying natural variation

Quantitative traits such as human height are measurable phenotypes that show continuous variation over a wide phenotypic range. Enormous effort has recently been put into determining the genetic influences on a variety of quantitative traits, including human genetic diseases, with mixed success. We identified a quantitative trait in a tractable model system, the GAL pathway in yeast, which controls the uptake and metabolism of the sugar galactose. GAL pathway activation depends both on galactose concentration and on the concentrations of competing, preferred sugars such as glucose. Natural yeast isolates show substantial variation in the behavior of the pathway. All studied yeast strains exhibit bimodal responses relative to external galactose concentration, i.e. a set of galactose concentrations existed at which both GAL-induced and GAL-repressed subpopulations were observed. However, these concentrations differed in different strains. We built a mechanistic model of the GAL pathway and identified parameters that are plausible candidates for capturing the phenotypic features of a set of strains including standard lab strains, natural variants, and mutants. In silico perturbation of these parameters identified variation in the intracellular galactose sensor, Gal3p, the negative feedback node within the GAL regulatory network, Gal80p, and the hexose transporters, HXT, as the main sources of the bimodal range variation. We were able to switch the phenotype of individual yeast strains in silico by tuning parameters related to these three elements. Determining the basis for these behavioral differences may give insight into how the GAL pathway processes information, and into the evolution of nutrient metabolism preferences in different strains. More generally, our method of identifying the key parameters that explain phenotypic variation in this system should be generally applicable to other quantitative traits. Author summaryMicrobes adopt elaborate strategies for the preferred uptake and use of nutrients to cope with complex and fluctuating environments. As a result, yeast strains originating from different ecological niches show significant variation in the way they induce genes in the galactose metabolism (GAL) pathway in response to nutrient signals. To identify the mechanistic sources of this variation, we built a mathematical model to simulate the dynamics of the galactose metabolic regulation network, and studied how parameters with different biological implications contributed to the natural variation. We found that variations in the behavior of the galactose sensor Gal3p, the negative feedback node Gal80p, and the hexose transporters HXT were critical elements in the GAL pathway response. Tuning single parameters in silico was sufficient to achieve phenotype switching between different yeast strains. Our computational approach should be generally useful to help pinpoint the genetic and molecular bases of natural variation in other systems.

systems biology↗

bioRxiv2020

Computational study on ratio-sensing in yeast galactose utilization pathway

Metabolic networks undergo gene expression regulation in response to external nutrient signals. In microbes, the synthesis of enzymes that are used to transport and catabolize less preferred carbon sources is repressed in the presence of a preferred carbon source. For most microbes, glucose is a preferred carbon source, and it has long been believed that as long as glucose is present in the environment, the expression of genes related to the metabolism of alternative carbon sources is shut down, due to catabolite repression. However, recent studies have shown that the induction of the galactose (GAL) metabolic network does not solely depend on the exhaustion of glucose. Instead, the GAL genes respond to the external concentration ratio of galactose to glucose, a phenomenon of unknown mechanism that we termed ratio-sensing. Using mathematical modeling, we found that ratio-sensing is a general phenomenon that can arise from competition between two carbon sources for shared transporters, between transcription factors for binding to communal regulatory sequences of the target genes, or a combination of the aforementioned two levels of competition. We analyzed how the parameters describing the competitive interaction influenced ratio-sensing behaviors in each scenario and found that the concatenation of both layers of signal integration can expand the dynamical range of ratio-sensing. Finally, we investigated the influence of circuit topology on ratio-sensing and found that incorporating negative auto-regulation and/or coherent feedforward loop motifs to the basic signal integration unit can tune the sensitivity of the response to the external nutrient signals. Our study not only deepened our understanding of how ratio-sensing is achieved in yeast GAL metabolic regulation, but also elucidated design principles for ratio-sensing signal processing that can be used in other biological settings, such as being introduced into circuit designs for synthetic biology applications. Author summaryMicrobes make sophisticated choices about the uptake and metabolism of nutrients depending on the variety of nutrient choices available to them in their environment. In the well-studied yeast galactose utilization network, a recent study has shown that galactose metabolic genes respond to the external concentration ratio of galactose to glucose. Using computational models, we showed that this type of phenomenon could arise from a competition between galactose and glucose for transporters, a competition between transcription factors for promoters, or a combination of these two mechanisms. We further revealed the controlling parameters that determined the system sensitivity towards competing input signals and that determined the concentration ratio required to induce the metabolic network in each scenario. Combining competition inhibition at both the transporter level and the transcriptional level can enlarge the ratio-sensing regime, resulting a robust signal integration module. We suspect that modules of this kind may be common in many areas of biology.

systems biology↗

bioRxiv2020

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

molecular biology↗

bioRxiv2019

Real-time monitoring of cotranscriptional riboswitch folding and switching

Riboswitches function through cotranscriptional conformation switching governed by cognate ligand concentration, RNA folding and transcription elongation kinetics. To investigate how these parameters influence riboswitch folding, we developed a novel vectorial folding assay (VF) in which the superhelicase Rep-X sequentially liberates the RNA strand from a heteroduplex in a 5-to-3 direction, mimicking the nascent chain emergence during transcription. The RNA polymerase (RNAP)-free VF recapitulates the kinetically controlled cotranscriptional folding of a ZTP riboswitch, whose activation is favored by slower transcription, strategic pausing, or a weakened transcriptional terminator. New methods to observe positions and local rates of individual helicases show an average Rep-X unwinding rate similar to bacterial RNAP elongation (~60 nt/s). Real-time single-molecule monitoring captured folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These methods allow observation of individual folding RNAs as they occupy distinct folding channels within the landscape that controls gene expression and showed that riboswitch fate control is encoded in its sequence and is readily interpreted by a directionally moving protein even in the absence of an RNA polymerase.

biophysics↗

bioRxiv2018

Circadian gene Clock regulates mitochondrial morphology and functions by posttranscriptional way

Many daily activities are under the control of circadian clock, including nutrition metabolism and energy generation. Mitochondria, as the core factories of oxidizing substrates and producing ATP, undergo changes in quantity and morphology to adapt to the demand for energy. It has been demonstrated that mitochondrial gene expression, dynamics and functions are all affected by circadian clock. Here, we demonstrated that circadian gene Clock affects the number, architecture and function of mitochondria via posttranscriptional regulation of Drp1. Clock{Delta}19 leads to fragmented mitochondria accompanied with the loss of membrane potential, excessive ROS accumulation and decreased mitochondrial respiration and ATP generation. Clock{Delta}19 mice exhibit disordered lipid metabolism and evident nonalcoholic fatty liver disease (NAFLD), which are rescued by treatment with the mitochondrial fission inhibitor Mdivi-1. These results suggest a strong relationship between Clock, mitochondrial dynamics and metabolic diseases and provide a new perspective on disordered circadian clock and related diseases.

cell biology↗

bioRxiv2017

Small effect-size mutations cumulatively affect yeast quantitative traits

SummaryQuantitative traits are influenced by pathways that have traditionally been defined through genes that have a large loss- or gain-of-function effect. However, in theory, a large number of small effect-size genes could cumulative play a substantial role in pathway function, potentially by acting as \"modifiers\" that tune the levels of large effect size pathway components. To understand the role of these small effect-size genes, we used a quantitative assay to determine the number, strength, and identity of all non-essential genes that affect two galactose-responsive (GAL) traits, in addition to re-analyzing two previously screened quantitative traits. Over a quarter of assayed genes have a detectable effect; approximately two thirds of the quantitative trait variation comes from small effect-size genes. The functions of small effect-size genes are partially overlapping between traits and are enriched in core cellular processes. This implies that genetic variation in one process has the potential to influence behavior or disease in seemingly unconnected processes.\n\nHighlightsO_LIFour yeast quantitative traits are affected by thousands of small effect-size genes.\nC_LIO_LISmall effect-size genes are enriched in core cellular processes\nC_LIO_LIThe effects of these genes are quantitative trait-specific.\nC_LI

systems biology↗

bioRxiv2017

Polymorphisms In The Yeast Galactose Sensor Underlie A Natural Continuum Of Nutrient-Decision Phenotypes

In nature, microbes often need to \"decide\" which of several available nutrients to utilize, a choice that depends on a cells inherent preference and external nutrient levels. While natural environments can have mixtures of different nutrients, phenotypic variation in microbes decisions of which nutrient to utilize is poorly studied. Here, we quantified differences in the concentration of glucose and galactose required to induce galactose-responsive (GAL) genes across 36 wild S. cerevisiae strains. Using bulk segregant analysis, we found that a locus containing the galactose sensor GAL3 was associated with differences in GAL signaling in eight different crosses. Using allele replacements, we confirmed that GAL3 is the major driver of GAL induction variation, and that GAL3 allelic variation alone can explain as much as 90% of the variation in GAL induction in a cross. The GAL3 variants we found modulate the diauxic lag, a selectable trait. These results suggest that ecological constraints on the galactose pathway may have led to variation in a single protein, allowing cells to quantitatively tune their response to nutrient changes in the environment.\n\nAuthor summaryIn nature, microbes often need to decide which of many potential nutrients to consume. This decision making process is complex, involving both intracellular constraints and the organisms perception of the environment. To begin to mimic the complexity of natural environments, we grew cells in mixtures of two sugars, glucose and galactose. We find that in mixed environments, the sugar concentration at which cells decides to induce galactose-utilizing (GAL) genes is highly variable in natural isolates of yeast. By analyzing crosses of phenotypically different strains, we identified a locus containing the galactose sensor, a gene that in theory could allow cells to tune their perception of the environment. We confirmed that the galactose sensor can explain upwards of 90% of the variation in the decision to induce GAL genes. Finally, we show that the variation in the galactose sensor can modulate the time required for cells to switch from utilizing glucose to galactose. Our results suggest that signaling pathways can be highly variable across strains and thereby might allow for rapid adaption in fluctuating environments.

genetics↗