Biology subjects

Roy, R.

Publications and source records attributed to Roy, R..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

AMPK regulates germline stem cell integrity and quiescence through a mir-1/tbc-7/rab-7 pathway in C. elegans.

During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as "dauer". During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that lack AMPK signalling, the germ cells fail to arrest, undergo uncontrolled proliferation and lose their reproductive capacity. These germline defects are accompanied by an altered chromatin landscape and gene expression program. We identified an allele of tbc-7, a RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is regulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. A singular small RNA, mir-1, was identified as a direct negative regulator of tbc-7 expression through the analysis of seed sequences on the 3UTR of tbc-7. Animals lacking mir-1 are post-dauer sterile, displaying a similar phenotype to AMPK mutants. Altogether, our findings describe a novel mir-1/tbc-7/rab-7 pathway occurring in the neurons that regulates the germ line in a cell non-autonomous manner.

genetics↗

bioRxiv2021

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

biochemistry↗

bioRxiv2021

Overriding defective FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in diabetic wound

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, extracted from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx (neutrophilia) as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the FPR1 chemokine receptor in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in wound in diabetic animals, and rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary chemokine receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR1 signaling and substantially improves infection control by jumpstarting the neutrophil response toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have real therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

immunology↗

bioRxiv2021

Convergent evolution of a novel blood-red nectar pigment in vertebrate-pollinated flowers

Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here we show that the nectars red color is derived from a novel alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including (1) a carbonic anhydrase that creates an alkaline environment, (2) an aryl alcohol oxidase that generates sinapaldehyde, a pigment precursor, and (3) a ferritin-like catalase that protects nesocodin from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a novel pigment with a stable imine bond, which in turn is attractive to Phelsuma geckos, the presumed pollinators of Nesocodon. We also identify nesocodin in the red nectar of the distantly related Jaltomata herrerae and provide evidence for convergent evolution of this trait. While the overall enzymatic activities required for red pigment formation in both Nesocodon and J. herrerae nectars are identical, the associated genes encoding the enzymes are not orthologous and, in the case of the aryl alcohol oxidase, even belong to different protein families. This work cumulatively identifies a novel, convergently evolved trait in two vertebrate-pollinated species, suggesting the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this adaptation. SignificanceNearly 90% of flowering plants produce nectar to attract pollinators. Beyond sugars, many types of nectar solutes play important ecological roles; however, the molecular basis for the diversity of nectar composition across species is less explored. One rare trait among flowering plants is the production of colored nectar, which may function to attract and guide prospective pollinators. Our findings indicate convergent evolution of a red-colored nectar across two distantly related plant species. Behavioral data show that the red pigment attracts diurnal geckos, a presumed pollinator of one of these plants. These findings join a growing list of examples of distinct biochemical and molecular mechanisms underlying evolutionary convergence, and provide a fascinating system for testing how interactions across species drive the evolution of novel pigments in an understudied context.

evolutionary biology↗

bioRxiv2021

RGG-motif protein Sbp1 is required for Processing body (P-body) disassembly

RNA granules are conserved mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. In this report, we identify RGG-motif translation repressor protein Sbp1 as a disassembly factor of P-bodies. Disassembly of Edc3 granules but not the Pab1 granules (a conserved stress granule marker) that arise upon sodium azide and glucose deprivation stress are defective in{Delta} sbp1. Disassembly of other P-body proteins such as Dhh1 and Scd6 is also defective in{Delta} sbp1. Complementation experiments suggest that the wild type Sbp1 but not an RGG-motif deletion mutant rescues the Edc3 granule disassembly defect in{Delta} sbp1. We observe that purified Edc3 forms assemblies, which is promoted by the presence of RNA and NADH. Strikingly, addition of purified Sbp1 leads to significantly decreased Edc3 assemblies. Although low complexity sequences have been in general implicated in assembly, our results reveal the role of RGG-motif (a low-complexity sequence) in the disassembly of P-bodies.

cell biology↗

bioRxiv2020

Schizophrenia and autism-associated mutations and disrupted m6A signal by YTHDF1 cause defects in microtubule function and neurodevelopment

Subcellular mRNA localization and local translation are crucial for spatially regulated gene expression in neurons. However, the precise mechanisms governing the selective transport and translation of mRNAs contributing to processes such as axonal growth and branching are only partially understood. Here, we present evidence of N6-methyladenosine (m6A)-mediated translational control of the RNA-binding protein, APC, influencing cytoskeletal dynamics at the growth cone. Our findings demonstrate that m6A modifications occur on Apc mRNA, facilitating its recognition by YTHDF1 to promote APC translation in neuronal somata. Additionally, we observe that disrupting the m6A pathway impairs the transport and local translation of {beta}-actin mRNA in the axon and growth cones, a deficiency that can be rescued by the exogenous expression of APC protein in cultured neurons. Furthermore, we establish the essential role of YTHDF1 in axon development, particularly in callosal projection neurons during cortical development. Our findings suggest a novel mechanism involving m6A-mediated regulation of APC protein translation, linking epitranscriptomics to axonal mRNA targeting, cytoskeletal dynamics, and axon development.

neuroscience↗

bioRxiv2020

PI3K inhibition as a novel therapeutic strategy for neoadjuvant chemoradiotherapy resistant oesophageal adenocarcinoma

ObjectivesNeoadjuvant chemo-radiotherapy (neo-CRT) prior to surgery is the standard of care for oesophageal adenocarcinoma (OAC) patients. Unfortunately, most patients fail to respond to treatment. MiR-187 was previously shown to be downregulated in neo-CRT non-responders, whist in vitro miR-187 overexpression enhanced radio-sensitivity and upregulated PTEN. This study evaluates the role of miR-187 and downstream PI3K signalling in radiation response in OAC. MethodsThe effect of miR-187 overexpression on downstream PI3K signalling was evaluated in OAC cell lines by qPCR and western blotting. PTEN expression was analysed in OAC pre-treatment biopsies of neo-CRT responders and non-responders. Pharmacological inhibition of PI3K using GDC-0941 was evaluated in combination with radiotherapy in 2D and 3D OAC models in vitro and as a single agent in vivo. Radiation response in vitro was assessed via clonogenic assay. ResultsPTEN expression was significantly decreased in neo-CRT non-responders. MiR-187 overexpression significantly upregulated PTEN expression and inhibited downstream PI3K signalling in vitro. GDC-0941 significantly reduced viability and enhanced radiation response in vitro and led to tumour growth inhibition as a single agent in vivo. ConclusionsTargeting of PI3K signalling is a promising therapeutic strategy for OAC patients who have repressed miR-187 expression and do not respond to conventional neo-CRT. Advances in knowledgeThis is the first study evaluating the effect of PI3K inhibition on radio-sensitivity in OAC, with a particular focus on patients that do not respond to neo-CRT. We have shown for the 1st time that targeting of PI3K signalling is a promising alternative therapeutic strategy for OAC patients who do not respond to conventional neo-CRT.

cancer biology↗

bioRxiv2020

BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1{+/-}0.2 and 1.24{+/-}0.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development,1, 2 and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.

immunology↗

bioRxiv2020

Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

Life cycle processes of positive-strand (+)RNA viruses are broadly conserved across families, yet they employ different life cycle strategies to grow in the cell. Using a generalized dynamical model for intracellular (+)ssRNA virus growth, we decipher these life cycle determinants and their dependencies for several viruses and parse the effect of viral mutations and host cell permissivity. We show that Poliovirus employs rapid replication and virus assembly whereas Japanese Encephalitis virus leverages its higher rate of translation and efficient cellular reorganization compared to Hepatitis C virus. Stochastic simulations of the model demonstrate infection extinction if all seeding viral RNA degrade before establishing robust replication. The probability of productive cellular infection is affected by virus-host processes, defined by early life cycle events and viral seeding. Synergy among these parameters in limiting infection suggests new avenues for inhibiting viral infections by targeting early life cycle bottlenecks.

microbiology↗

bioRxiv2020

Translation regulation of Japanese encephalitis virus revealed by ribosome profiling

Japanese encephalitis virus (JEV), a neurotropic flavivirus, is the leading cause of viral encephalitis in endemic regions of Asia. Although the mechanisms modulating JEV virulence and neuroinvasiveness are poorly understood, several acquired mutations in the live attenuated vaccine strain (SA14-14-2) point towards translation regulation as a key strategy. Using ribosome profiling, we identify multiple mechanisms including frameshifting, tRNA dysregulation and alternate translation initiation sites that regulate viral protein synthesis. A significant fraction (~ 40%) of ribosomes undergo frameshifting on NS1 coding sequence leading to early termination, translation of NS1' protein and modulation of viral protein stoichiometry. Separately, a tRNA subset (glutamate, serine, leucine and histidine) was found to be associated in high levels with the ribosomes upon JEV infection. We also report a previously uncharacterised translational initiation event from an upstream UUG initiation codon in JEV 5' UTR. A silent mutation at this start site in the vaccine strain has been shown to abrogate neuroinvasiveness suggesting the potential role of translation from this region. Together, our study sheds light on distinct mechanisms that modulate JEV translation with likely consequences for viral pathogenesis.

microbiology↗

bioRxiv2020

A Peptide of the Amino-Terminus of GRK2 Induces Hypertrophy and Yet Elicits Cardioprotection after Pressure Overload

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 “interactome” that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. While elevated cardiac levels and activity of GRK2 contribute to adverse heart remodeling and contractile dysfunction, inhibition of GRK2 via overexpression of a carboxyl-terminal peptide, βARKct, or its amino-terminal domain Regulator of G protein Signaling (RGS) homology domain (βARKrgs) can enhance cardiac function and can prevent heart failure development via Gβγ or Gαq sequestration, respectively. Previously, our lab investigated cardiac-specific transgenic expression of a fragment of this RGS domain (βARKnt) (residues 50-145). In contrast to βARKrgs this fragment did not alter acute hypertrophy after pressure overload or demonstrate RGS activity in vivo against Gq-mediated signaling. Herein, we subjected these transgenic mice to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, βARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, βARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. These data support the idea that the βARKnt peptide embodies a distinct functional interaction and novel means of cardioprotection during pressure-overload induced heart failure.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGPCRG protein-coupled receptor;GRK2G protein-coupled receptor kinase 2;RGSregulator of G protein Signaling domain;βARbeta-adrenergic receptor;LVleft ventricular;TACtransverse aortic constriction;αMHCα-myosin heavy chain;NLCnon-transgenic littermate control;WGAwheat germ agglutinin;IPimmunoprecipitation;IP3Inositol 1,4,5-trisphosphate;125I-CYP[125I]cyanopindolol;HFpEFheart failure with preserved ejection fraction;HFrEFheart failure with reduced ejection fraction;InsRinsulin receptor;IRSinsulin receptor substrate;PI3Kphosphoinositide 3-kinase;PDK1 and 2phosphatidylinositol-dependent kinases;PKB/Aktprotein kinase B;AS160Akt substrate of 160 kilodaltons;GAPGTPase-activating protein;GSK3βglycogen synthase kinase 3β.View Full Text

physiology↗

bioRxiv2020

An RNA dynamic ensemble at atomic resolution

Biomolecules do not fold into a single 3D structure but rather form dynamic ensembles of many inter-converting conformations1. Knowledge of dynamic ensembles is key for understanding how biomolecules fold and function, and for rationally manipulating their activities in drug discovery and synthetic biology2-4. However, solving dynamic ensembles of biomolecules at atomic resolution is a major challenge in structural biology because the information required to specify the position of all atoms in thousands of conformations in an ensemble far exceeds the information content of experimental measurements. Here we addressed the data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformational library. Library refinement with NMR residual dipolar couplings enabled determination of an atomic-resolution ensemble for HIV-1 TAR as confirmed by quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through the successful redistribution of the ensemble by design using atomic mutagenesis. The ensemble provides an unprecedented view of how bulge residues cooperatively flip out and undergo sugar repuckering to allow the adjoining helices to stack. The generality of this approach will make determination of atomic-resolution RNA ensembles routine.

biophysics↗

bioRxiv2019

Systems analyses of key metabolic modules of floral and extrafloral nectaries of cotton

Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. In Gossypium hirsutum (cotton), four distinct trichomatic nectaries develop, one floral and three extrafloral. The secreted floral and extrafloral nectars serve different purposes, with the floral nectar attracting bees to promote pollination and the extrafloral nectar attracting predatory insects as a means of indirect resistance from herbivores. Cotton therefore provides an ideal system to contrast mechanisms of nectar production and nectar composition between floral and extrafloral nectaries. Here, we report the transcriptome, ultrastructure, and metabolite spatial distribution using mass spectrometric imaging of the four cotton nectary types throughout development. Additionally, the secreted nectar metabolomes were defined and were jointly composed of 197 analytes, 60 of which were identified. Integration of theses datasets support the coordination of merocrine-based and eccrine-based models of nectar synthesis. The nectary ultrastructure supports the merocrine-based model due to the abundance of rough endoplasmic reticulum positioned parallel to the cell walls and profusion of vesicles fusing to the plasma membranes. The eccrine-based model which consist of a progression from starch synthesis to starch degradation and to sucrose biosynthesis was supported by gene expression data. This demonstrates conservation of the eccrine-based model for the first time in both trichomatic and extrafloral nectaries. Lastly, nectary gene expression data provided evidence to support de novo synthesis of amino acids detected in the secreted nectars. One sentence summaryThe eccrine-based model of nectar synthesis and secretion is conserved in both trichomatic and extrafloral nectaries determined by a system-based comparison of cotton (Gossypium hirsutum) nectaries.

plant biology↗

bioRxiv2019

Metabolically-active obligate aerobes in anoxic (sulfidic) marine sediments

Metabolically-active obligate aerobes are unheard-of in tightly-anoxic environments. Present culture-independent and culture-dependent investigations revealed aerobic microbial communities along two, ~3-meter-long sediment-cores underlying the eastern Arabian Sea oxygen minimum zone, where high H2S disallows O2 influx from the water-column. While genes for aerobic respiration by aa3-/cbb3-type cytochrome-c oxidases and cytochrome-bd ubiquinol oxidase, and aerobic oxidation of methane/ammonia/alcohols/thiosulfate/sulfite/organosulfur-compounds, were present across the cores, so were live aerobic, sulfur-chemolithoautotrophs and chemoorganoheterotrophs. The 8820-years-old, highly-sulfidic, methane-containing sediment-sample from 275 cmbsf of 530 mbsl yielded many such obligately-aerobic bacterial-isolates that died upon anaerobic incubation with alternative electron-acceptors/fermentative-substrates. Several metatranscriptomic reads from this sediment-sample matched aerobic-respiration-/oxidase-reaction-/transcription-/translation-/DNA-replication-/membrane-transport-/cell-division-related genes of the obligately-aerobic isolates, thereby corroborating their active aerobic metabolic-status in situ. Metagenomic and metatranscriptomic detection of perchlorate-/chlorate-reduction genes, plus anaerobic growth of an obligately-aerobic Halothiobacillus isolate in the presence of perchlorate and perchlorate-reducing-consortia, suggested that cryptic O2 produced by perchlorate-respirers could be sustaining obligately-aerobes in this environment.

microbiology↗

bioRxiv2019

Activation or inhibition of PPARα-mediated fatty acid β-oxidation does not active cardiomyocyte proliferation in normal or infarcted adult mice

ObjectivesPPAR genes are known as the important regulators of fatty acid oxidation and energy homeostasis. PPAR is highly expressed in the embryonic and adult heart. Previous studies from infant mouse hearts have suggested that activation of PPAR using GW7647 treatment or cardiac-restricted activation of PPAR using MHC-PPAR transgenic mice enhanced fatty acid {beta}-oxidation and promoted cardiomyocyte proliferation rate in the postnatal day 4 mouse heart. Here, we further investigate the impact of PPAR-mediated fatty acid {beta}-oxidation on cardiomyocyte proliferation in the adult mouse heart.\n\nMethods and ResultsAdult wild-type (C57BL/6J) mice were subjected to five injections of GW7647, a highly specific PPAR agonist, or vehicle (saline). Cardiomyocyte proliferation was analyzed by quantification of DNA synthesis via ethynyldeoxyuridine (EdU) incorporation and quantification of cells undergoing mitosis using phosphorylated histone H3 (PH3). GW7647 treatment resulted in activation of PPAR target genes associated with fatty acid metabolism and {beta}-oxidation, validating its biological activity. However, GW7647 treatment did not active cardiomyocyte proliferation in the normal heart. In parallel, mice were subjected to myocardial infarction (MI) using permanent coronary artery occlusion. Both GW7647-treatd wild-type mice and MHC-PPAR transgenic mice showed no significant differences in cardiomyocyte DNA synthesis and mitosis compared with vehicle-treated wild-type mice after MI. Furthermore, inhibition of PPAR-mediated fatty acid {beta}-oxidation using etomoxir (ETO) treatment had no impact on cardiomyocyte proliferation in both normal and infarcted hearts of wild-type mice compared with vehicle treatment.\n\nSummaryThese findings suggest that activation or inhibition of PPAR-mediated fatty acid {beta}-oxidation did not active cardiomyocyte proliferation in normal or infarcted hearts of adult mice. Any effects on cardiac function observed following PPAR activation treatment is independent of enhanced cardiomyocyte renewal in the adult heart.

cell biology↗

bioRxiv2018

An integrated transcriptomics and metabolomics analysis of the Cucurbita pepo nectary implicates key modules of primary metabolism involved in nectar synthesis and secretion

Nectar is the main reward that flowers offer to pollinators to entice repeated visitation. Cucurbita pepo (squash) is an excellent model for studying nectar biology, as it has large nectaries that produce large volumes of nectar relative to most other species. Squash is also monoecious, having both female and male flowers on the same plant, which allows comparative analyses of nectary function in one individual. Here we report the nectary transcriptomes from both female and male nectaries at four stages of floral maturation. Analysis of these transcriptomes and subsequent confirmatory experiments revealed a metabolic progression in nectaries leading from starch synthesis to starch degradation and to sucrose biosynthesis. These results are consistent with previously published models of nectar secretion and also suggest how a sucrose-rich nectar can be synthesized and secreted in the absence of active transport across the plasma membrane. Non-targeted metabolomic analyses of nectars also confidently identified 40 metabolites in both female and male nectars, with some displaying preferential accumulation in nectar of either male or female flowers. Cumulatively, this study identified gene targets for reverse genetics approaches to study nectary function, as well as previously unreported nectar metabolites that may function in plant-biotic interactions.

plant biology↗

bioRxiv2018

TH2BS12P histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes

Various studies have focussed on understanding the repertoire and biological function of the post-translational modifications that occur on testis-specific histone variants like TH2B, Transition Proteins etc. In our attempt to decipher the unique functions of histone variant TH2B, we discovered a new modification Serine 12 phosphorylation on TH2B (TH2BS12P) in spermatocytes. Our present study is aimed at understanding the function of the TH2BS12P modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies revealed that TH2BS12P histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body axes associated proteins like Scp3, {gamma}H2AX, pATM, ATR etc. We also observe that TH2BS12P is associated with DSB initiator Spo11 and with several recombination related proteins like pATM, ATR, Rad51, {gamma}H2AX etc in vivo. This modification was also found to associate with transcription and recombination related histone marks like H3K4me3 and H3K36me3 in the context of mononucleosomes. Genome-wide occupancy studies as determined by ChIP sequencing experiments revealed that TH2BS12P is localised to subset of recombination hotspots, but majorly associated with H3K4me3 containing genomic regions like gene promoters. Mass spectrometry analysis of proteins that bind to TH2BS12P containing mononucleosomes revealed many proteins linked with the functions of pericentric heterochromatin, transcription and recombination related pathways. We propose that TH2BS12P modification could act alone or in concert with other histone marks for recruitment of appropriate transcription or recombination protein machinery at specific genomic loci. This is the first report documenting the role of a post-translational modification of a germ cell specific histone variant in meiotic prophase I related events.

cell biology↗

bioRxiv2018

AMPK regulates germline stem cell quiescence and integrity through an endogenous small RNA pathway.

C. elegans larvae can undergo a global developmental arrest following the execution of a diapause-like state called dauer in response to unfavourable growth conditions. Survival in this stage surpasses the normal lifespan of reproductive animals quite dramatically, and without any apparent negative impact on their reproductive fitness. During this period, the germ cells become quiescent and must retain their reproductive integrity so the animal can reproduce following recovery. This germline stem cell (GSC) arrest requires the activity of AMP-activated protein kinase (AMPK) and in its absence the germ line undergoes hyperplasia. We show here that AMPK mutant animals exhibit complete sterility after recovery from dauer, suggesting that germ cell integrity is compromised during this stage in the absence of AMPK. These defects correlate with altered abundance and distribution of a number of chromatin modifications that affect gene expression. These aberrant chromatin modifications, along with the supernumerary germ cell divisions and the observed post-dauer sterility, were all corrected by disabling key effectors of the small interfering RNA pathway (dcr-1 and rde-4) and the primary Argonaute protein ergo-1, suggesting that AMPK regulates the function of these small RNA pathway components, and in its absence, the pathways become abnormally active. The aberrant regulation of the small RNA pathway components releases the germ cells from quiescence to proliferative state thereby compromising germ cell integrity. Curiously, AMPK expression in either the neurons or the excretory system is sufficient to restore the GSC quiescence and the fertility in the AMPK mutant post-dauer adults, while the fertility of these animals is also partially restored by disabling the dsRNA importer SID-1. Our data suggest that AMPK regulates a small RNA pathway in the soma to establish and/or maintain GSC quiescence and integrity cell non-autonomously in response to the energy stress associated with the dauer stage. Our findings therefore provide a unique model to better understand how the soma communicates with the germ line to establish the appropriate epigenetic modifications required to adapt to acute environmental challenges.

developmental biology↗