Epigenetic variation creates potential for evolution of plant phenotypic plasticity

Heritable variation in plant phenotypes, and thus potential for evolutionary change, can in principle not only be caused by variation in DNA sequence, but also by underlying epigenetic variation. However, the potential scope of such phenotypic effects and their evolutionary significance are largely unexplored. Here, we conducted a glasshouse experiment in which we tested the response of a large number of epigenetic recombinant inbred lines (epiRILs) of Arabidopsis thaliana – lines that are nearly isogenic but highly variable at the level of DNA methylation – to drought and increased nutrient conditions. We found significant heritable variation among epiRILs both in the means of several ecologically important plant traits and in their plasticities to drought and nutrients. Significant selection gradients, that is, fitness correlations, of several mean traits and plasticities suggest that selection could act on this epigenetically based phenotypic variation. Our study provides evidence that variation in DNA methylation can cause substantial heritable variation of ecologically important plant traits, including root allocation, drought tolerance and nutrient plasticity, and that rapid evolution based on epigenetic variation alone should thus be possible.

IDH/MGMT-driven molecular classification of low-grade glioma is a strong predictor for long-term survival

BACKGROUND Low-grade gliomas (LGGs) are rare brain neoplasms, with survival spanning up to a few decades. Thus, accurate evaluations on how biomarkers impact survival among patients with LGG require long-term studies on samples prospectively collected over a long period. METHODS The 210 adult LGGs collected in our databank were screened for IDH1 and IDH2 mutations (IDHmut), MGMT gene promoter methylation (MGMTmet), 1p/19q loss of heterozygosity (1p19qloh), and nuclear TP53 immunopositivity (TP53pos). Multivariate survival analyses with multiple imputation of missing data were performed using either histopathology or molecular markers. Both models were compared using Akaike's information criterion (AIC). The molecular model was reduced by stepwise model selection to filter out the most critical predictors. A third model was generated to assess for various marker combinations. RESULTS Molecular parameters were better survival predictors than histology (ΔAIC = 12.5, P< .001). Forty-five percent of studied patients died. MGMTmet was positively associated with IDHmut (P< .001). In the molecular model with marker combinations, IDHmut/MGMTmet combined status had a favorable impact on overall survival, compared with IDHwt (hazard ratio [HR] = 0.33, P< .01), and even more so the triple combination, IDHmut/MGMTmet/1p19qloh (HR = 0.18, P< .001). Furthermore, IDHmut/MGMTmet/TP53pos triple combination was a significant risk factor for malignant transformation (HR = 2.75, P< .05). CONCLUSION By integrating networks of activated molecular glioma pathways, the model based on genotype better predicts prognosis than histology and, therefore, provides a more reliable tool for standardizing future treatment strategies.

Arsenic volatilization in model anaerobic biogas digesters

Arsenic is a class 1 non-threshold carcinogen which is highly ubiquitous. Arsenic undergoes many different transformations (biotic or abiotic) between and within environmental compartments, leading to a number of different chemical species possessing different properties and toxicities. One specific transformation is As biotic volatilization which is coupled with As biomethylation and has been scarcely studied due to inherent sampling issues. Arsenic methylation/volatilization is also linked with methanogenesis and occurs in anaerobic environments. In China, rice straw and animal manure are very often used to produce biogas and both can contain high amounts of As, especially if the rice is grown in areas with heavy mining or smelting industries and if Roxarsone is fed to the animals. Roxarsone is an As-containing drug which is widely used in China to control coccidian intestinal parasites, to improve feed efficiency and to promote rapid growth. Previous work has shown that this compound degrades to inorganic As under anaerobic conditions. In this study the focus is on biotic transformations of As in small microcosms designed as biogas digester models (BDMs) using recently validated As traps, thus, enabling direct quantification and identification of volatile As species. It is shown that although there was a loss of soluble As in the BDMs, their conditions favored biomethylation. All reactors produced volatile As, especially the monomethylarsonic acid spiked ones with 413 ± 148 ng As (mean ± SD, n = 3) which suggest that the first methylation step, from inorganic As, is a limiting factor. The most abundant species was trimethylarsine, but the toxic arsine was present in the headspace of most of the BDMs. The results suggest that volatile As species should be monitored in biogas digesters in order to assess risks to humans working in biogas plants and those utilizing the biogas.

Isotope fractionation of selenium by biomethylation in microcosm incubations of soil

The natural abundance of stable Se isotopes in methylselenides reflects sources and formation conditions of methylselenides. We tested the effects of (i) different inorganic Se species spiked to soils and (ii) different soil samples on the extent of fungal biomethylation of Se and the Se isotope ratios (δ82/76Se) in methylselenides. Furthermore, we assessed the decrease of dissolved, bioavailable Se during three days of equilibration of the soils with Se-enriched solutions. We conducted closed microcosm experiments containing soil spiked with Se(IV) or Se(VI), a growth medium, and the fungus species Alternaria alternata for 11 d. The concentrations and isotope ratios of Se were determined in all components of the microcosm with multicollector ICP-MS. The equilibration of the spiked Se(IV) and Se(VI) for 3 d resulted in a decrease of dissolved, bioavailable Se concentrations by 32 to 44% and 8 to 14%, respectively. Very little isotope fractionation occurred during this phase, and it can be attributed to mixing of the added Se with the pre-existing Se in the soils and minor Se(IV) reduction in one experiment. In two of the incubated soils – moderately acidic roadside and garden soils – between 9.1 and 30% of the supplied Se(IV) and 1.7% of the supplied Se(VI) were methylated while in a strongly acidic forest soil no Se methylation occurred. The methylselenides derived from Se(IV) were strongly depleted in 82Se (δ82/76Se = − 3.3 to − 4.5‰) compared with the soil (0.16–0.45‰) and the added Se(IV) (0.20‰). The methylselenide yield of the incubations with Se(VI) was too small for isotope measurements. Our results demonstrate that Se source species and soil properties influence the extent of Se biomethylation and that the produced methylselenides contain isotopically light Se.

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

Fetal programming of pulmonary vascular dysfunction in mice: role of epigenetic mechanisms.

Insults during the fetal period predispose the offspring to systemic cardiovascular disease, but little is known about the pulmonary circulation and the underlying mechanisms. Maternal undernutrition during pregnancy may represent a model to investigate underlying mechanisms, because it is associated with systemic vascular dysfunction in the offspring in animals and humans. In rats, restrictive diet during pregnancy (RDP) increases oxidative stress in the placenta. Oxygen species are known to induce epigenetic alterations and may cross the placental barrier. We hypothesized that RDP in mice induces pulmonary vascular dysfunction in the offspring that is related to an epigenetic mechanism. To test this hypothesis, we assessed pulmonary vascular function and lung DNA methylation in offspring of RDP and in control mice at the end of a 2-wk exposure to hypoxia. We found that endothelium-dependent pulmonary artery vasodilation in vitro was impaired and hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in vivo were exaggerated in offspring of RDP. This pulmonary vascular dysfunction was associated with altered lung DNA methylation. Administration of the histone deacetylase inhibitors butyrate and trichostatin A to offspring of RDP normalized pulmonary DNA methylation and vascular function. Finally, administration of the nitroxide Tempol to the mother during RDP prevented vascular dysfunction and dysmethylation in the offspring. These findings demonstrate that in mice undernutrition during gestation induces pulmonary vascular dysfunction in the offspring by an epigenetic mechanism. A similar mechanism may be involved in the fetal programming of vascular dysfunction in humans.

Origins of Shared Genetic Variation in African Cichlids

Cichlid fishes have evolved tremendous morphological and behavioral diversity in the waters of East Africa. Within each of the Great Lakes Tanganyika, Malawi, and Victoria, the phenomena of hybridization and retention of ancestral polymorphism explain allele sharing across species. Here, we explore the sharing of single nucleotide polymorphisms (SNPs) between the major East African cichlid assemblages. A set of approximately 200 genic and nongenic SNPs was ascertained in five Lake Malawi species and genotyped in a diverse collection of 160 species from across Africa. We observed segregating polymorphism outside of the Malawi lineage for more than 50% of these loci; this holds similarly for genic versus nongenic SNPs, as well as for SNPs at putative CpG versus non-CpG sites. Bayesian and principal component analyses of genetic structure in the data demonstrate that the Lake Malawi endemic flock is not monophyletic and that river species have likely contributed significantly to Malawi genomes. Coalescent simulations support the hypothesis that river cichlids have transported polymorphism between lake assemblages. We observed strong genetic differentiation between Malawi lineages for approximately 8% of loci, with contributions from both genic and nongenic SNPs. Notably, more than half of these outlier loci between Malawi groups are polymorphic outside of the lake. Cichlid fishes have evolved diversity in Lake Malawi as new mutations combined with standing genetic variation shared across East Africa.

MEN1 Gene Mutation and Reduced Expression Are Associated With Poor Prognosis in Pulmonary Carcinoids

Context: MEN1 gene alterations have been implicated in lung carcinoids, but their effect on gene expression and disease outcome is unknown. Objective: Our objective was to analyze MEN1 gene and expression anomalies in lung neuroendocrine neoplasms and their correlations with clinicopathologic data and disease outcome. Design: We examined 74 lung neuroendocrine neoplasms including 58 carcinoids and 16 high-grade neuroendocrine carcinomas (HGNECs) for MEN1 mutations (n = 70) and allelic losses (n = 69), promoter hypermethylation (n = 65), and mRNA (n = 74) expression. Results were correlated with disease outcome. Results: MEN1 mutations were found in 7 of 55 (13%) carcinoids and in 1 HGNEC, mostly associated with loss of the second allele. MEN1 decreased expression levels correlated with the presence of mutations (P = .0060) and was also lower in HGNECs than carcinoids (P = .0024). MEN1 methylation was not associated with mRNA expression levels. Patients with carcinoids harboring MEN1 mutation and loss had shorter overall survival (P = .039 and P = .035, respectively) and low MEN1 mRNA levels correlated with distant metastasis (P = .00010) and shorter survival (P = .0071). In multivariate analysis, stage and MEN1 allelic loss were independent predictors of prognosis. Conclusion: Thirteen percent of pulmonary carcinoids harbor MEN1 mutation associated with reduced mRNA expression and poor prognosis. Also in mutation-negative tumors, low MEN1 gene expression correlates with an adverse disease outcome. Hypermethylation was excluded as the underlying mechanism.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the "serrated pathway" characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases. Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out. Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34-0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and - deficient cancers was 87% [OR (95% CI): 0.96 (0.95-0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases. Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events "upstream" of the development of microsatellite instability may impact Cdx2 expression.

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Watson–Crick and Sugar-Edge Base Pairing of Cytosine in the Gas Phase: UV and Infrared Spectra of Cytosine·2-Pyridone

While keto-amino cytosine is the dominant species in aqueous solution, spectroscopic studies in molecular beams and in noble gas matrices show that other cytosine tautomers prevail in apolar environments. Each of these offers two or three H-bonding sites (Watson–Crick, wobble, sugar-edge). The mass- and isomer-specific S1 ← S0 vibronic spectra of cytosine·2-pyridone (Cyt·2PY) and 1-methylcytosine·2PY are measured using UV laser resonant two-photon ionization (R2PI), UV/UV depletion, and IR depletion spectroscopy. The UV spectra of the Watson–Crick and sugar-edge isomers of Cyt·2PY are separated using UV/UV spectral hole-burning. Five different isomers of Cyt·2PY are observed in a supersonic beam. We show that the Watson–Crick and sugar-edge dimers of keto-amino cytosine with 2PY are the most abundant in the beam, although keto-amino-cytosine is only the third most abundant tautomer in the gas phase. We identify the different isomers by combining three different diagnostic tools: (1) methylation of the cytosine N1–H group prevents formation of both the sugar-edge and wobble isomers and gives the Watson–Crick isomer exclusively. (2) The calculated ground state binding and dissociation energies, relative gas-phase abundances, excitation and the ionization energies are in agreement with the assignment of the dominant Cyt·2PY isomers to the Watson–Crick and sugar-edge complexes of keto-amino cytosine. (3) The comparison of calculated ground state vibrational frequencies to the experimental IR spectra in the carbonyl stretch and NH/OH/CH stretch ranges strengthen this identification.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-A density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.