SEARCH · Search bioRxiv

Results for “Biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 919 records · Page 51Linked to original sources

bioRxiv2018

Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of open conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP+ and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP+ and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP+ shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a conformational lockdown model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes.\n\nA note to the readerThis manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.

biochemistry↗

bioRxiv2018

Active site closure stabilizes the backtracked state of RNA polymerase

All cellular RNA polymerases (RNAP) occasionally backtrack along the template DNA as part of transcriptional proofreading and regulation. Here, we studied the mechanism of RNAP backtracking by one nucleotide using two complementary approaches that allowed us to precisely measure the occupancy and lifetime of the backtracked state. Our data show that the stability of the backtracked state is critically dependent on the closure of the RNAP active site by a mobile domain, the trigger loop (TL). The lifetime and occupancy of the backtracked state measurably decreased by substitutions of the TL residues that interact with the nucleoside triphosphate (NTP) substrate, whereas amino acid substitutions that stabilized the closed active site increased the lifetime and occupancy. These results suggest that the same conformer of the TL closes the active site during catalysis of nucleotide incorporation into the nascent RNA and backtracking by one nucleotide. In support of this hypothesis, we construct a model of the 1-nt backtracked complex with the closed active site and the backtracked nucleotide in the entry pore area known as the E-site. We further propose that 1-nt backtracking mimics the reversal of the NTP substrate loading into the RNAP active site during on-pathway elongation.

biochemistry↗

bioRxiv2018

IMPROVING THE PERFORMANCE OF THE RNA AMBER FORCE FIELD BY TUNING THE HYDROGEN-BONDING INTERACTIONS

Molecular dynamics (MD) simulations became a leading tool for investigation of structural dynamics of nucleic acids. Despite recent efforts to improve the empirical potentials (force fields, ffs), RNA ffs have persisting deficiencies, which hamper their utilization in quantitatively accurate simulations. Previous studies have shown that at least two salient problems contribute to difficulties in description of free-energy landscapes of small RNA motifs: (i) excessive stabilization of the unfolded single-stranded RNA ensemble by intramolecular base-phosphate and sugar-phosphate interactions, and (ii) destabilization of the native folded state by underestimation of stability of base pairing. Here, we introduce a general ff term (gHBfix) that can selectively fine-tune non-bonding interaction terms in RNA ffs, in particular the H-bonds. gHBfix potential affects the pair-wise interactions between all possible pairs of the specific atom types, while all other interactions remain intact, i.e., it is not a structure-based model. In order to probe the ability of the gHBfix potential to refine the ff non-bonded terms, we performed an extensive set of folding simulations of RNA tetranucleotides and tetraloops. Based on these data we propose particular gHBfix parameters to modify the AMBER RNA ff. The suggested parametrization significantly improves the agreement between experimental data and the simulation conformational ensembles, although our current ff version still remains far from being flawless. While attempts to tune the RNA ffs by conventional reparametrizations of dihedral potentials or non-bonded terms can lead to major undesired side effects as we demonstrate for some recently published ffs, gHBfix has a clear promising potential to improve the ff performance while avoiding introduction of major new imbalances.

biochemistry↗

bioRxiv2018

Effect of Sec61 interaction with Mpd1 on Endoplasmic Reticulum-Associated Degradation

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we identified new lumenal interaction partners of Sec61 by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.

biochemistry↗

bioRxiv2018

Developing a unique medical-grade honey which maximizes glucose oxidase activity

In the fight against cancer and infection, honey is a compelling solution by virtue of its unique chemical composition; however, current medical-grade honeys are expensive and limited in their scope. In this study, a novel medical-grade honey was developed by maximizing the activity of glucose oxidase (GOX), an enzyme in honey that synthesizes hydrogen peroxide; maximization was done by neutralizing the effects of catalase and methylglyoxal (MGO), compounds in honey that interfere with H2O2 accumulation. Expressed GOX activity was quantified via H2O2 accumulation in honey after catalase, MGO, or both were inhibited. Results indicate the honeys tested have significant quantities of H2O2 inhibitors, greatly affecting expressed GOX activity; neutralization resulted in at least a 100% increase in H2O2 accumulation in all honeys. Blueberry honey with catalase inhibition by EGCG saw a 938% increase in H2O2 accumulation, reaching nearly three times the H2O2 accumulation in current medical grade honey for a tenth of the cost. This research presents a novel and readily reproducible method for maximizing H2O2 accumulation in any honey through neutralization of GOX inhibitors. GOX activity enhancement, combined with honeys diverse antioxidants, enables the emergence of global low-cost medical-grade honeys with immense potential to revolutionize cancer and infection treatment.\n\nThis paper is based on work presented at the Regional Science and Engineering Fair on March 15, 2018 at Riverside High School (Loudoun County, Virginia), the Virginia State Science and Engineering Fair on April 14, 2018 at the Virginia Tech Carilion School of Medicine (Roanoke, Virginia).\n\nAuthor SummaryWith cancer and post-operative infections on the rise, a natural, low-cost alternative treatment is necessary. Honey is an ideal candidate due to its diverse antioxidants and glucose oxidase, an enzyme that produces hydrogen peroxide. The purpose of this research was to maximize glucose oxidase activity by neutralizing compounds that interfere with H2O2 accumulation. This research yielded medical-grade honeys that are economical and more effective than current ones, presenting a novel method that is reproducible on a large scale, scalable to different needs, and applicable to any honey to create low-cost medical-grade honeys that can revolutionize cancer and infection treatment globally.

biochemistry↗

bioRxiv2018

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase

DiSulfide Bond (DSB) oxidative folding enzymes are master regulators of virulence localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K12 has a dithiol oxidizing redox relay pair (DsbA/B), a disulfide isomerizing redox relay pair (DsbC/D) and specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these -DsbA1 and -DsbB form a redox relay pair analogous to E. coli DsbA/B. The third enzyme -DsbA2 incorporates a DsbA-like sequence but does not interact with -DsbB. In comparison with other DsbA enzymes, -DsbA2 has [~]50 extra N-terminal residues. The crystal structure of -DsbA2{Delta}N, the N-terminally truncated form in which these residues are removed confirms the DsbA-like nature of this domain. However, -DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. First, -DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase - ie its role is to shuffle rather than introduce disulfide bonds. Moreover, small-angle X-ray scattering of -DsbA2 reveals a homotrimeric arrangement. Our results allow us to draw conclusions about the factors required for functionally equivalent enzymatic activity across structurally diverse protein architectures.

biochemistry↗

bioRxiv2018

Management of Hsp90-Dependent Protein Folding by Small Molecule Targeting the Aha1 Co-Chaperone

The core cytosolic Hsp90 chaperone/co-chaperone complex plays a critical role in proteostasis management of human health and disease. To identify novel compounds that alter the ability of the Hsp90 co-chaperone Aha1 to modulate the ATPase activity found in multiple folding diseases ranging from steroid hormone receptor (SHR) sensitive prostate cancer to tauopathies associated with neurodegenerative diseases, we employed a high throughput screening (HTS) assay to monitor selectively Aha1-stimulated Hsp90 (ASH) ATPase activity. The ASH assay identified SEW04784 (SEW), a small molecule that disrupts ASH activity without inhibiting the basal Hsp90 ATPase activity. NMR analysis reveals that SEW binds to the C-terminal domain of Aha1 to disrupt its asymmetric binding to Hsp90 leading to abrogation of its chaperoning activity of Hsp90. SEW exhibits therapeutic potential by blocking the transcriptional activity of prostate cancer (PCa) associated variants of the androgen receptor (AR) in a cell-based model of PCa. Additionally, SEW exhibits the ability to clear toxic, phosphorylated tau aggregated species associated with tauopathies. By not directly impacting the basal ATPase function of the abundant and ubiquitous Hsp90, SEW could provide a therapeutic approach for mitigation of client-specific proteostatic disease.

biochemistry↗

bioRxiv2018

Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5 part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

biochemistry↗

bioRxiv2018

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming a tripartite complex that acts as a competitive antagonist of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of this complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.

biochemistry↗

bioRxiv2018

Structural basis of light-induced redox regulation in the Calvin cycle

In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.\n\nOne Sentence SummaryHow plants turn off carbon fixation in the dark.

biochemistry↗

bioRxiv2018

The biochemical basis of microRNA targeting efficacy

MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of mRNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA-target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute-miRNA complexes and all [≤]12-nucleotide sequences. This approach revealed noncanonical target sites unique to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.

biochemistry↗

bioRxiv2018

Regulation of Histone Deacetylase 3 by Metal Cations and 10-Hydroxy-2E-Decenoic Acid: Possible Molecular Epigenetic Mechanisms of Queen-Worker Bee Differentiation

Histone deacetylases (HDACs) catalyze the hydrolysis of {varepsilon}-acetyl-lysine residues of histones. Removal of acetyl groups results in condensation of chromatin structure and repression of gene expression. Human class I, II, and IV HDACs are said to be zinc-dependent in that they require divalent zinc ions to catalyze the deacetylase reaction. HDACs are considered potential targets for the treatment of cancer due to their role in regulating transcription. They are also thought to play important roles in the development of organisms such as honey bees. The fatty acid, 10-hydroxy-2E-decenoic acid (10-HDA), which can account for up to 5% of royal jelly composition has been reported as an HDAC inhibitor. The crystal structure of the HDAC3:SMRT complex possesses two monovalent cations (MVCs) labeled as potassium with one MVC binding site near the active site Zn(II) and the second MVC binding site [≥]20 [A] from the active site Zn(II). We report here the inhibitory effects of excess Zn(II) on the catalytic activity of histone deacetylase 3 (HDAC3) bound to the deacetylase activating domain of nuclear receptor corepressor 2 (NCOR2). We also report the effects of varying concentrations of potassium ions where [K+] up to 10 mM increase HDAC3 activity with a maximum kcat/KM of approximately 80,000 M-1s-1 while [K+] above 10 mM inhibit HDAC3 activity. The inhibition constant (Ki) of 10-HDA was determined to be 5.32 mM. The regulatory effects of zinc, potassium, and 10-HDA concentration on HDAC3 activity suggest a strong correlation between these chemical species and epigenetic control over Apis mellifera caste differentiation among other control mechanisms.

biochemistry↗

bioRxiv2018

The metabolic repair enzyme phospho-glycolate phosphatase regulates central carbon metabolism and fosmidomycin sensitivity in Plasmodium falciparum

The asexual blood stages of the malaria parasite, Plasmodium falciparum are highly dependent on glycolysis for ATP synthesis, redox balance and provision of essential anabolic precursors. Recent studies have suggested that members of the haloacid dehalogenase (HAD) family of metabolite phosphatases may play an important role in regulating multiple pathways in P. falciparum central carbon metabolism. Here, we show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PfPGP), which is homologous to yeast Pho13 and mammalian PGP, regulates glycolysis in asexual blood stages by controlling intracellular levels of several intermediates and novel end-products of this pathway. Deletion of the P. falciparum pgp gene significantly attenuated asexual parasite growth in red blood cells, while comprehensive metabolomic analysis revealed the accumulation of two previously uncharacterized metabolites, as well as changes in a number of intermediates in glycolysis and the pentose phosphate pathway. The two unknown metabolites were assigned as 2-phospho-lactate and 4-phosphoerythronate by comparison of their mass spectra with synthetic standards. 2-Phospho-lactate was significantly elevated in wildtype and {Delta}PfPGP parasites cultivated in the presence of methylglyoxal and D-lactate, but not L-lactate, indicating that it is a novel end-product of the methylglyoxal pathway. 4-Phosphoerythronate is a putative side product of the glycolytic enzyme, glyceraldehyde dehydrogenase and the accumulation of both 4-phosphoerythronate and 2-phospho-D-lactate were associated with changes in glycolytic and the pentose phosphate pathway fluxes as shown by 13C-glucose labelling studies and increased sensitivity of the {Delta}PfPGP parasites to the drug fosmidomycin. Our results suggest that PfPGP contributes to a novel futile metabolic cycle involving the phosphorylation/dephosphorylation of D-lactate as well as detoxification of metabolites, such as 4-phosphoerythronate, and both may have important roles in regulating P. falciparum central carbon metabolism.\n\nAuthor summaryThe major pathogenic stages of the malaria parasite, Plasmodium falciparum, develop in red blood cells where they have access to an abundant supply of glucose. Unsurprisingly these parasite stages are addicted to using glucose, which is catabolized in the glycolytic and the pentose phosphate pathways. While these pathways also exist in host cells, there is increasing evidence that P. falciparum has evolved novel ways for regulating glucose metabolism that could be targeted by next-generation of anti-malarial drugs. In this study, we show the red blood cell stages of P. falciparum express an enzyme that is specifically involved in regulating the intracellular levels of two metabolites that are novel end-products or side products of glycolysis. Parasite mutants lacking this enzyme are viable but exhibit diminished growth rates in red blood cells. These mutant lines accumulate the two metabolites, and exhibit global changes in central carbon metabolism. Our findings suggest that metabolic end/side products of glycolysis directly regulate the metabolism of these parasites, and that the intracellular levels of these are tightly controlled by previously uncharacterized metabolite phosphatases.

biochemistry↗

bioRxiv2018

The molecular structure of plant sporopollenin

Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains. Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered as a prerequisite for the migration of early plants onto land. Despite its importance, the chemical structure of plant sporopollenin has remained elusive. Using a newly developed thioacidolysis degradative method together with state-of-the-art solid-state NMR techniques, we determined the detailed molecular structure of pine sporopollenin. We show that pine sporopollenin is primarily composed of aliphatic-polyketide-derived polyvinyl alcohol units and 7-O-p-coumaroylated C16 aliphatic units, crosslinked through a distinctive m-dioxane moiety featuring an acetal. Naringenin was also identified as a minor component of pine sporopollenin. This discovery answers the long-standing question about the chemical makeup of plant sporopollenin, laying the foundation for future investigations of sporopollenin biosynthesis and for design of new biomimetic polymers with desirable inert properties.

biochemistry↗

bioRxiv2018

A novel interaction between Gβγ and RNA polymerase II regulates cardiac fibrosis.

G{beta}{gamma} subunits are involved in many different signalling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear G{beta}{gamma}, we investigated the functional role of G{beta}{gamma} signalling in regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. Following activation of the angiotensin II type I receptor in these cells, G{beta}{gamma} dimers interact with RNA polymerase II (RNAPII). Our findings suggest that G{beta}1 recruitment to RNAPII negatively regulates the fibrotic transcriptional response, which can be overcome by strong fibrotic stimuli. The interaction between G{beta}{gamma} subunits and RNAPII expands the role for G{beta}{gamma} signalling in cardiac fibrosis. The G{beta}{gamma}-RNAPII interaction was regulated by signaling pathways in HEK 293 cells that diverged from those operating in cardiac fibroblasts. Thus, the interaction may be a conserved feature of transcriptional regulation although such regulation may be cell-specific.

biochemistry↗

bioRxiv2018

The Synergistic Effect of Antioxidant Interaction between Luteolin and Chlorogenic Acid in Lonicera japonica

Lonicera japonica Thunb. is a flower that is used in traditional Chinese medicine to prevent the common cold. The two primary active compounds of the flower bud are luteolin, a flavonoid, and chlorogenic acid, a phenolic acid. Both active compounds have demonstrated antioxidant activity. The interactions between chemicals in a plant heavily influences its total antioxidant activity. We attempted to investigate the antioxidant interactions between the two chemicals in the plant. This study aims to investigate if the antioxidants luteolin and chlorogenic acid have a synergistic effect to inhibit free radicals when combined. A 2,2-diphenyl-1-picrylhydrazyl (DPPH*) assay was performed. The half maximal inhibitory concentration (IC50) of luteolin and chlorogenic acid were first determined and then combined at a 1:1 ratio. The combined inhibition capacity was then compared with the sum of the individual inhibition capacities. The IC50 of luteolin is 26.304 g{middle dot}ml-1 {+/-} 0.120 g{middle dot}ml-1 while the IC50 of chlorogenic acid is 85.529 g{middle dot}ml-1 {+/-} 4.482 g{middle dot}ml-1. The combined solution produced a free radical percentage inhibition of 77.617% {+/-} 5.470%, more than the percentage inhibition of the separate solutions. The experiment shows that luteolin and chlorogenic acid have a synergistic effect in inhibiting DPPH free radicals.

biochemistry↗

bioRxiv2018

Visualizing Deep Mutational Scan Data

Site-directed and random mutagenesis are biochemical tools to obtain insights into the structure and function of proteins. Recent advances such as deep mutational scan have allowed a complete scan of all the amino acid positions in a protein with each of the 19 possible alternatives. Mapping out the phenotypic consequences of thousands of single point mutations in the same protein is now possible. Visualizing and analysing the rich data offers an opportunity to learn more about the effects of mutations, for a better understanding and engineering of proteins. This work focuses on such visualization analyses applied to the mutational data of TEM-1 {beta}-lactamase. The data is examined in the light of the expected biochemical effects of single point mutations, with the goal of reinforcing or retraining the intuitions. Individual attributes of the amino acid mutations such as the solvent accessible area, charge type change, and distance from the catalytic center capture most of the relevant functional effects. Visualizing the data suggests how combinations of these attributes can be used for a better classification of the effects of mutations, when independently they do not offer a high predictability.

biochemistry↗

bioRxiv2018

Precise re-deposition of nucleosomes on repressive chromatin domains sustain epigenetic inheritance during DNA replication

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.

biochemistry↗