RIF1 regulates early replication timing in murine B cells.

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated, hypermutating murine B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin and promotes early replication, but plays a minor role in regulating replication origin activity, gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal additional layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination.

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S) sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3' CTCF-binding element (3'CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR.

N-glycan antennal modifications are altered in Caenorhabditis elegans lacking the HEX-4 N-acetylgalactosamine-specific hexosaminidase.

Simple organisms are often considered to have simple glycomes, but plentiful paucimannosidic and oligomannosidic glycans overshadow the less abundant N-glycans with highly variable core and antennal modifications; Caenorhabditis elegans is no exception. By use of optimized fractionation and assessing wildtype in comparison to mutant strains lacking either the HEX-4 or HEX-5 ß-N-acetylgalactosaminidases, we conclude that the model nematode has a total N-glycomic potential of 300 verified isomers. Three pools of glycans were analyzed for each strain: either PNGase F released and eluted from a reversed-phase C18 resin with either water or 15% methanol or PNGase Ar released. While the water-eluted fractions were dominated by typical paucimannosidic and oligomannosidic glycans and the PNGase Ar-released pools by glycans with various core modifications, the methanol-eluted fractions contained a huge range of phosphorylcholine-modified structures with up to three antennae, sometimes with four N-acetylhexosamine residues in series. There were no major differences between the C. elegans wildtype and hex-5 mutant strains, but the hex-4 mutant strains displayed altered sets of methanol-eluted and PNGase Ar-released pools. In keeping with the specificity of HEX-4, there were more glycans capped with N-acetylgalactosamine in the hex-4 mutants, as compared with isomeric chito-oligomer motifs in the wildtype. Considering that fluorescence microscopy showed that a HEX-4::enhanced GFP fusion protein colocalizes with a Golgi tracker, we conclude that HEX-4 plays a significant role in late-stage Golgi processing of N-glycans in C. elegans. Furthermore, finding more "parasite-like" structures in the model worm may facilitate discovery of glycan-processing enzymes occurring in other nematodes.

DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations.

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.

Glycomics, Glycoproteomics, and Glycogenomics: An Inter-Taxa Evolutionary Perspective.

Glycosylation is a highly diverse set of co- and posttranslational modifications of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue-, and species-specific and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological, and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes-different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.

Zwitterionic Phosphodiester-Substituted Neoglycoconjugates as Ligands for Antibodies and Acute Phase Proteins.

Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds. Furthermore, in array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs. Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals.

Tissue-specific glycosylation in the honeybee: Analysis of the N-glycomes of Apis mellifera larvae and venom.

BACKGROUND: Previous glycophylogenetic comparisons of dipteran and lepidopteran species revealed variations in the anionic and zwitterionic modifications of their N-glycans; therefore, we wished to explore whether species- and order-specific glycomic variations would extend to the hymenoptera, which include the honeybee Apis mellifera, an agriculturally- and allergologically-significant social species. METHODS: In this study, we employed an off-line liquid chromatography/mass spectrometry approach, in combination with enzymatic and chemical treatments, to analyse the N-glycans of male honeybee larvae and honeybee venom in order to facilitate definition of isomeric structures. RESULTS: The neutral larval N-glycome was dominated by oligomannosidic and paucimannosidic structures, while the neutral venom N-glycome displayed more processed hybrid and complex forms with antennal N-acetylgalactosamine, galactose and fucose residues including Lewis-like epitopes; the anionic pools from both larvae and venom contained a wide variety of glucuronylated, sulphated and phosphoethanolamine-modified N-glycans with up to three antennae. In comparison to honeybee royal jelly, there were more fucosylated and fewer Man4/5-based hybrid glycans in the larvae and venom samples as well as contrasting antennal lengths. CONCLUSIONS: Combining the current data on venom and larvae with that we previously published on royal jelly, a total honeybee N-glycomic repertoire of some 150 compositions can be proposed in addition to the 20 previously identified on specific venom glycoproteins. SIGNIFICANCE: Our data are indicative of tissue-specific modification of the core and antennal regions of N-glycans in Apis mellifera and reinforce the concept that insects are capable of extensive processing to result in rather complex anionic oligosaccharide structures.

Protein-Specific Analysis of Invertebrate Glycoproteins.

N-Glycans are posttranslational modifications of proteins attached to the amide side chains of asparagine residues, with possible heterogeneity due to different structures being possible at the same glycosylation site. In contrast to the mammalian systems, invertebrate N-glycosylation presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species. A simple analytical approach to analyze N-glycans on specific glycoproteins is presented, which involves a combination of tryptic peptide mass spectrometry and "off-line" RP-HPLC MALDI-TOF MS/MS complemented by blotting to recognize specific epitopes. An additional N-glycan enrichment and labeling step can facilitate the analysis of single structures and even provide isomeric separation of N-glycans from specific proteins.

Isomeric Separation and Recognition of Anionic and Zwitterionic N-glycans from Royal Jelly Glycoproteins.

Royal jelly has received attention because of its necessity for the development of queen honeybees as well as claims of benefits on human health; this product of the hypopharyngeal glands of worker bees contains a large number of proteins, some of which have been claimed to have various biological effects only in their glycosylated state. However, although there have been glycomic and glycoproteomic analyses in the past, none of the glycan structures previously defined would appear to have potential to trigger specific biological functions. In the current study, whole royal jelly as well as single protein bands were subject to off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting and arraying data. Similarly to recent in-depth studies on other insect species, previously overlooked glucuronic acid termini, sulfation of mannose residues and core ß-mannosylation of the N-glycans were found; additionally, a relatively rare zwitterionic modification with phosphoethanolamine is present, in contrast to the phosphorylcholine occurring in lepidopteran species. Indicative of tissue-specific remodelling of glycans in the Golgi apparatus of hypopharyngeal gland cells, only a low amount of fucosylated or paucimannosidic glycans were detected as compared with other insect samples or even bee venom. The unusual modifications of hybrid and multiantennary structures defined here may not only have a physiological role in honeybee development, but represent epitopes recognized by pentraxins with roles in animal innate immunity.

Hydrophilic interaction anion exchange for separation of multiply modified neutral and anionic Dictyostelium N-glycans.

The unusual nature of the N-glycans of the cellular slime mould Dictyostelium discoideum has been revealed by a number of studies, primarily based on examination of radiolabeled glycopeptides but more recently also by MS. The complexity of the N-glycomes of even glycosylation mutants is compounded by the occurrence of anionic modifications, which also present an analytical challenge. In this study, we have employed hydrophilic interaction anion exchange (HIAX) HPLC in combination with MALDI-TOF MS/MS to explore the anionic N-glycome of the M31 (modA) strain, which lacks endoplasmic reticulum α-glucosidase II, an enzyme conserved in most eukaryotes including Homo sapiens. Prefractionation with HIAX chromatography enabled the identification of N-glycans with unusual oligo-α1,2-mannose extensions as well as others with up to four anionic modifications. Due to the use of hydrofluoric acid treatment, we were able to discriminate isobaric glycans differing in the presence of sulphate or phosphate on intersected structures as opposed to those carrying GlcNAc-phosphodiesters. The latter represent biosynthetic intermediates during the pathway leading to formation of the methylphosphorylated mannose epitope, which may have a similar function in intracellular targeting of hydrolases as the mannose-6-phosphate modification of lysosomal enzymes in mammals. In conclusion, HIAX in combination with MS is a highly sensitive approach for both fine separation and definition of neutral and anionic N-glycan structures.

The underestimated N-glycomes of lepidopteran species.

BACKGROUND: Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability. METHODS: Using an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line. RESULTS: We detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed. CONCLUSION: The lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species. SIGNIFICANCE: The occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.