CD4+ T cell immunity is dependent on an intrinsic stem-like program.

CD4+ T cells are central to various immune responses, but the molecular programs that drive and maintain CD4+ T cell immunity are not entirely clear. Here we identify a stem-like program that governs the CD4+ T cell response in transplantation models. Single-cell-transcriptomic analysis revealed that naive alloantigen-specific CD4+ T cells develop into TCF1hi effector precursor (TEP) cells and TCF1-CXCR6+ effectors in transplant recipients. The TCF1-CXCR6+CD4+ effectors lose proliferation capacity and do not reject allografts upon adoptive transfer into secondary hosts. By contrast, the TCF1hiCD4+ TEP cells have dual features of self-renewal and effector differentiation potential, and allograft rejection depends on continuous replenishment of TCF1-CXCR6+ effectors from TCF1hiCD4+ TEP cells. Mechanistically, TCF1 sustains the CD4+ TEP cell population, whereas the transcription factor IRF4 and the glycolytic enzyme LDHA govern the effector differentiation potential of CD4+ TEP cells. Deletion of IRF4 or LDHA in T cells induces transplant acceptance. These findings unravel a stem-like program that controls the self-renewal capacity and effector differentiation potential of CD4+ TEP cells and have implications for T cell-related immunotherapies.

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting.

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

CDK7 regulates organ size and tumor growth by safeguarding the Hippo pathway effector Yki/Yap/Taz in the nucleus.

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.

Correcting PCR amplification errors in unique molecular identifiers to generate accurate numbers of sequencing molecules.

Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.

Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model.

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

Expression of the monocarboxylate transporter MCT1 is required for virus-specific mouse CD8+ T cell memory development.

Lactate-proton symporter monocarboxylate transporter 1 (MCT1) facilitates lactic acid export from T cells. Here, we report that MCT1 is mandatory for the development of virus-specific CD8+ T cell memory. MCT1-deficient T cells were exposed to acute pneumovirus (pneumonia virus of mice, PVM) or persistent γ-herpesvirus (Murid herpesvirus 4, MuHV-4) infection. MCT1 was required for the expansion of virus-specific CD8+ T cells and the control of virus replication in the acute phase of infection. This situation prevented the subsequent development of virus-specific T cell memory, a necessary step in containing virus reactivation during γ-herpesvirus latency. Instead, persistent active infection drove virus-specific CD8+ T cells toward functional exhaustion, a phenotype typically seen in chronic viral infections. Mechanistically, MCT1 deficiency sequentially impaired lactic acid efflux from activated CD8+ T cells, caused an intracellular acidification inhibiting glycolysis, disrupted nucleotide synthesis in the upstream pentose phosphate pathway, and halted cell proliferation which, ultimately, promoted functional CD8+ T cell exhaustion instead of memory development. Taken together, our data demonstrate that MCT1 expression is mandatory for inducing T cell memory and controlling viral infection by CD8+ T cells.

A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Entanglement-based secure quantum cryptography over 1,120 kilometres.

Quantum key distribution (QKD)1-3 is a theoretically secure way of sharing secret keys between remote users. It has been demonstrated in a laboratory over a coiled optical fibre up to 404 kilometres long4-7. In the field, point-to-point QKD has been achieved from a satellite to a ground station up to 1,200 kilometres away8-10. However, real-world QKD-based cryptography targets physically separated users on the Earth, for which the maximum distance has been about 100 kilometres11,12. The use of trusted relays can extend these distances from across a typical metropolitan area13-16 to intercity17 and even intercontinental distances18. However, relays pose security risks, which can be avoided by using entanglement-based QKD, which has inherent source-independent security19,20. Long-distance entanglement distribution can be realized using quantum repeaters21, but the related technology is still immature for practical implementations22. The obvious alternative for extending the range of quantum communication without compromising its security is satellite-based QKD, but so far satellite-based entanglement distribution has not been efficient23 enough to support QKD. Here we demonstrate entanglement-based QKD between two ground stations separated by 1,120 kilometres at a finite secret-key rate of 0.12 bits per second, without the need for trusted relays. Entangled photon pairs were distributed via two bidirectional downlinks from the Micius satellite to two ground observatories in Delingha and Nanshan in China. The development of a high-efficiency telescope and follow-up optics crucially improved the link efficiency. The generated keys are secure for realistic devices, because our ground receivers were carefully designed to guarantee fair sampling and immunity to all known side channels24,25. Our method not only increases the secure distance on the ground tenfold but also increases the practical security of QKD to an unprecedented level.

Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Electrostatic-induced green and precise growth of model catalysts.

Crystallographic control of crystals as catalysts with precise geometrical and chemical features is significantly important to develop sustainable chemistry, yet highly challenging. Encouraged by first principles calculations, precise structure control of ionic crystals could be realized by introducing an interfacial electrostatic field. Herein, we report an efficient in situ dipole-sourced electrostatic field modulation strategy using polarized ferroelectret, for crystal facet engineering toward challenging catalysis reactions, which avoids undesired faradic reactions or insufficient field strength by conventional external electric field. Resultantly, a distinct structure evolution from tetrahedron to polyhedron with different dominated facets of Ag3PO4 model catalyst was obtained by tuning the polarization level, and similar oriented growth was also realized by ZnO system. Theoretical calculations and simulation reveal that the generated electrostatic field can effectively guide the migration and anchoring of Ag+ precursors and free Ag3PO4 nuclei, achieving oriented crystal growth by thermodynamic and kinetic balance. The faceted Ag3PO4 catalyst exhibits high performance in photocatalytic water oxidation and nitrogen fixation for valuable chemicals production, validating the effectiveness and potential of this crystal regulation strategy. Such an electrically tunable growth concept by electrostatic field provides new synthetic insights and great opportunity to effectively tailor the crystal structures for facet-dependent catalysis.

Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation.

In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.

The lysine deacetylase activity of histone deacetylases 1 and 2 is required to safeguard zygotic genome activation in mice and cattle.

The genome is transcriptionally inert at fertilization and must be activated through a remarkable developmental process called zygotic genome activation (ZGA). Epigenetic reprogramming contributes significantly to the dynamic gene expression during ZGA; however, the mechanism has yet to be resolved. Here, we find histone deacetylases 1 and 2 (HDAC1/2) can regulate ZGA through lysine deacetylase activity. Notably, in mouse embryos, overexpression of a HDAC1/2 dominant-negative mutant leads to developmental arrest at the two-cell stage. RNA-seq reveals that 64% of downregulated genes are ZGA genes and 49% of upregulated genes are developmental genes. Inhibition of the deacetylase activity of HDAC1/2 causes a failure of histone deacetylation at multiple sites, including H4K5, H4K16, H3K14, H3K18 and H3K27. ChIP-seq analysis exhibits an increase and decrease of H3K27ac enrichment at promoters of up- and downregulated genes, respectively. Moreover, HDAC1 mutants prohibit the removal of H3K4me3 by impeding expression of Kdm5 genes. Importantly, the developmental block can be greatly rescued by Kdm5b injection and by partially correcting the expression of the majority of dysregulated genes. Similar functional significance of HDAC1/2 is conserved in bovine embryos. Overall, we propose that HDAC1/2 are indispensable for ZGA by creating correct transcriptional repressive and active states in mouse and bovine embryos.

Hypoxia-driven tumor stromal remodeling and immunosuppressive microenvironment in scirrhous HCC.

BACKGROUND AND AIMS: Scirrhous HCC (SHCC) is one of the unique subtypes of HCC, characterized by abundant fibrous stroma in the tumor microenvironment. However, the molecular traits of SHCC remain unclear, which is essential to develop specialized therapeutic approaches for SHCC. APPROACH AND RESULTS: We presented an integrative analysis containing single-cell RNA-sequencing, whole-exome sequencing, and bulk RNA-sequencing in SHCC and usual HCC samples from 134 patients to delineate genomic features, transcriptomic profiles, and stromal immune microenvironment of SHCC. Multiplexed immunofluorescence staining, flow cytometry, and functional experiments were performed for validation. Here, we identified SHCC presented with less genomic heterogeneity while possessing a unique transcriptomic profile different from usual HCC. Insulin-like growth factor 2 was significantly upregulated in SHCC tumor cells compared to usual HCC, and could serve as a potential diagnostic biomarker for SHCC. Significant tumor stromal remodeling and hypoxia were observed in SHCC with enrichment of matrix cancer-associated fibroblasts and upregulation of hypoxic pathways. Insulin-like growth factor 2 was identified as a key mediator in shaping the hypoxic stromal microenvironment of SHCC. Under this microenvironment, SHCC exhibited an immunosuppressive niche correlated to enhanced VEGFA signaling activity, where CD4 + T cells and CD8 + T cells were dysfunctional. Furthermore, we found that another hypoxic-related molecule SPP1 from SHCC tumor cells suppressed the function of dendritic cells via the SPP1-CD44 axis, which also probably hindered the activation of T cells. CONCLUSION: We uncovered the genomic characteristics of SHCC, and revealed a hypoxia-driven tumor stroma remodeling and immunosuppressive microenvironment in SHCC.

Weissella paramesenteroides NRIC1542 inhibits dextran sodium sulfate-induced colitis in mice through regulating gut microbiota and SIRT1/NF-κB signaling pathway.

Inflammatory bowel disease (IBD) is a kind of recurrent inflammatory disorder of the intestinal tract. The purpose of this study was to investigate the effects of Weissella paramesenteroides NRIC1542 on colitis in mice. A colitis model was induced by adding 1.5% DSS to sterile distilled water for seven consecutive days. During this process, mice were administered different concentrations of W. paramesenteroides NRIC1542. Colitis was assessed by DAI, colon length and hematoxylin-eosin staining of colon sections. The expressions of NF-κB signaling proteins and the tight junction proteins ZO-1 and occludin were detected by western blotting, and the gut microbiota was analyzed by 16S rDNA. The results showed that W. paramesenteroides NRIC1542 significantly reduced the degree of pathological tissue damage and the levels of TNF-α and IL-1ß in colonic tissue, inhibiting the NF-κB signaling pathway and increasing the expression of SIRT1, ZO-1 and occludin. In addition, W. paramesenteroides NRIC1542 can modulate the structure of the gut microbiota, characterized by increased relative abundance of Muribaculaceae_unclassified, Paraprevotella, Prevotellaceae_UCG_001 and Roseburia, and decrease the relative abundance of Akkermansia and Alloprevotella induced by DSS. The above results suggested that W. paramesenteroides NRIC1542 can protect against DSS-induced colitis in mice through anti-inflammatory, intestinal barrier maintenance and flora modulation.

Podocan promoting skeletal muscle post-injury regeneration by inhibiting TGF-ß signaling pathway.

Podocan, the fifth member of Small Leucine-Rich Proteoglycan (SLRP) family of extracellular matrix components, is poorly known in muscle development. Previous studies have shown that Podocan promotes C2C12 differentiation in mice. In this study, we elucidated the effect of Podocan on skeletal muscle post-injury regeneration and its underlying mechanism. Injection of Podocan protein promoted the process of mice skeletal muscle post-injury regeneration. This effect seemed to be from the acceleration of muscle satellite cell differentiation in vivo. Meanwhile, Podocan promoted myogenic differentiation in vitro by binding with TGF-ß1 to inhibit the activity of the TGF-ß signaling pathway. These results indicated that Podocan had the potential roles to enhance skeletal muscle post-injury regeneration. Its mechanism is likely the regulation of the expression of p-Smad2 and p-Smad4 related to the TGF-ß signaling pathway by interacting with TGF-ß1.

The battle between host antiviral innate immunity and immune evasion by cytomegalovirus.

Cytomegalovirus (CMV) has successfully established a long-lasting latent infection in humans due to its ability to counteract the host antiviral innate immune response. During coevolution with the host, the virus has evolved various evasion techniques to evade the host's innate immune surveillance. At present, there is still no vaccine available for the prevention and treatment of CMV infection, and the interaction between CMV infection and host antiviral innate immunity is still not well understood. However, ongoing studies will offer new insights into how to treat and prevent CMV infection and its related diseases. Here, we update recent studies on how CMV evades antiviral innate immunity, with a focus on how CMV proteins target and disrupt critical adaptors of antiviral innate immune signaling pathways. This review also discusses some classic intrinsic cellular defences that are crucial to the fight against viral invasion. A comprehensive review of the evasion mechanisms of antiviral innate immunity by CMV will help investigators identify new therapeutic targets and develop vaccines against CMV infection.

Stabilizing microbial communities by looped mass transfer.

Building and changing a microbiome at will and maintaining it over hundreds of generations has so far proven challenging. Despite best efforts, complex microbiomes appear to be susceptible to large stochastic fluctuations. Current capabilities to assemble and control stable complex microbiomes are limited. Here, we propose a looped mass transfer design that stabilizes microbiomes over long periods of time. Five local microbiomes were continuously grown in parallel for over 114 generations and connected by a loop to a regional pool. Mass transfer rates were altered and microbiome dynamics were monitored using quantitative high-throughput flow cytometry and taxonomic sequencing of whole communities and sorted subcommunities. Increased mass transfer rates reduced local and temporal variation in microbiome assembly, did not affect functions, and overcame stochasticity, with all microbiomes exhibiting high constancy and increasing resistance. Mass transfer synchronized the structures of the five local microbiomes and nestedness of certain cell types was eminent. Mass transfer increased cell number and thus decreased net growth rates µ'. Subsets of cells that did not show net growth µ'SCx were rescued by the regional pool R and thus remained part of the microbiome. The loop in mass transfer ensured the survival of cells that would otherwise go extinct, even if they did not grow in all local microbiomes or grew more slowly than the actual dilution rate D would allow. The rescue effect, known from metacommunity theory, was the main stabilizing mechanism leading to synchrony and survival of subcommunities, despite differences in cell physiological properties, including growth rates.

Intestinal precursors avoid being misinduced to liver cells by activating Cdx-Wnt inhibition cascade.

During coordinated development of two neighboring organs from the same germ layer, how precursors of one organ resist the inductive signals of the other to avoid being misinduced to wrong cell fate remains a general question in developmental biology. The liver and anterior intestinal precursors located in close proximity along the gut axis represent a typical example. Here we identify a zebrafish leberwurst (lbw) mutant with a unique hepatized intestine phenotype, exhibiting replacement of anterior intestinal cells by liver cells. lbw encodes the Cdx1b homeoprotein, which is specifically expressed in the intestine, and its precursor cells. Mechanistically, in the intestinal precursors, Cdx1b binds to genomic DNA at the regulatory region of secreted frizzled related protein 5 (sfrp5) to activate sfrp5 transcription. Sfrp5 blocks the mesoderm-derived, liver-inductive Wnt2bb signal, thus conferring intestinal precursor cells resistance to Wnt2bb. These results demonstrate that the intestinal precursors avoid being misinduced toward hepatic lineages through the activation of the Cdx1b-Sfrp5 cascade, implicating Cdx/Sfrp5 as a potential pharmacological target for the manipulation of intestinal-hepatic bifurcations, and shedding light on the general question of how precursor cells resist incorrect inductive signals during embryonic development.

Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency.

The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice.

Evolutionary conservation of sequence motifs at sites of protein modification.

Gene duplications are common in biology and are likely to be an important source of functional diversification and specialization. The yeast Saccharomyces cerevisiae underwent a whole-genome duplication event early in evolution, and a substantial number of duplicated genes have been retained. We identified more than 3500 instances where only one of two paralogous proteins undergoes posttranslational modification despite having retained the same amino acid residue in both. We also developed a web-based search algorithm (CoSMoS.c.) that scores conservation of amino acid sequences based on 1011 wild and domesticated yeast isolates and used it to compare differentially modified pairs of paralogous proteins. We found that the most common modifications-phosphorylation, ubiquitylation, and acylation but not N-glycosylation-occur in regions of high sequence conservation. Such conservation is evident even for ubiquitylation and succinylation, where there is no established 'consensus site' for modification. Differences in phosphorylation were not associated with predicted secondary structure or solvent accessibility but did mirror known differences in kinase-substrate interactions. Thus, differences in posttranslational modification likely result from differences in adjoining amino acids and their interactions with modifying enzymes. By integrating data from large-scale proteomics and genomics analysis, in a system with such substantial genetic diversity, we obtained a more comprehensive understanding of the functional basis for genetic redundancies that have persisted for 100 million years.