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BACKGROUND: The prevalence of fungi in cystic fibrosis (CF) lung infections is poorly understood and studies have focused on adult patients. We investigated the fungal diversity in children with CF using bronchoalveolar lavage (BAL) and induced sputum (IS) samples to capture multiple lung niches. METHODS: Sequencing of the fungal ITS2 region and molecular mycobiota diversity analysis was performed on 25 matched sets of BAL-IS samples from 23 children collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). RESULTS: Aspergillus and Candida were detected in all samples and were the most abundant and prevalent genera, followed by Dipodascus, Lecanicillium and Simplicillium. The presumptive CF pathogens Exophiala, Lomentospora and Scedosporium were identified at variable abundances in 100 %, 64 %, and 24 % of sample sets, respectively. Fungal pathogens observed at high relative abundance (≥40 %) were not accurately diagnosed by routine culture microbiology in over 50 % of the cohort. The fungal communities captured by BAL and IS samples were similar in diversity and composition, with exception to C. albicans being significantly increased in IS samples. The respiratory mycobiota varied greatly between individuals, with only 13 of 25 sample sets containing a dominant fungal taxon. In 11/25 BAL sample sets, airway compartmentalisation was observed with diverse mycobiota detected from different lobes of the lung. CONCLUSIONS: The paediatric mycobiota is diverse, complex and inadequately diagnosed by conventional microbiology. Overlapping fungal communities were identified in BAL and IS samples, showing that IS can capture fungal genera associated with the lower airway. Compartmentalisation of the lower airway presents difficulties for consistent mycobiota sampling.
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Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.
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Proteínas de Drosophila , Complexo Repressor Polycomb 1 , Animais , Sistema Nervoso Central/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Larva/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.
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Adenina , Metilação de DNA , Proteínas de Drosophila , Drosophila , Animais , Cromatografia Líquida , DNA/genética , Drosophila/genética , Espectrometria de Massas em TandemRESUMO
DNMT3 in Hymenoptera has a unique duplication of the essential PWWP domain. Using GST-tagged PWWP fusion proteins and histone arrays we show that these domains have gained new properties and represent the first case of PWWP domains binding to H3K27 chromatin modifications, including H3K27me3, a key modification that is important during development. Phylogenetic analyses of 107 genomes indicate that the duplicated PWWP domains separated into two sister clades, and their distinct binding capacities are supported by 3D modeling. Other features of this unique DNA methylation system include variable copies, losses, and duplications of DNMT1 and DNMT3, and combinatorial generations of DNMT3 isoforms including variants missing the catalytic domain. Some of these losses and duplications of are found only in parasitic wasps. We discuss our findings in the context of the crosstalk between DNA methylation and histone methylation, and the expanded potential of epigenomic modifications in Hymenoptera to drive evolutionary novelties.
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Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred â¼1â h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.
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Cálcio , Timerosal , Camundongos , Masculino , Animais , Timerosal/farmacologia , Sêmen , Espermatozoides/fisiologia , Trifosfato de Adenosina , Fertilização/fisiologiaRESUMO
The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiologia , Águas Residuárias , Pandemias , Vigilância Epidemiológica Baseada em Águas Residuárias , Testes ImediatosRESUMO
Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.
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Doença Pulmonar Obstrutiva Crônica , Transcriptoma , Humanos , Epigênese Genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Pulmão/patologia , Perfilação da Expressão Gênica , Metilação de DNARESUMO
Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.
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Diabetes Mellitus , Células Secretoras de Glucagon , Camundongos , Animais , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Insulina/metabolismo , Células Secretoras de Glucagon/metabolismo , Metilação de DNA , Diabetes Mellitus/metabolismoRESUMO
TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.
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5-Metilcitosina , Dioxigenases , 5-Metilcitosina/metabolismo , Animais , Domínio Catalítico , Fenômenos Fisiológicos Celulares , DNA , Dioxigenases/genética , Dioxigenases/metabolismo , Mamíferos/genéticaRESUMO
Aging is characterized by a chronic low-grade inflammation known as inflammaging in multiple tissues, representing a risk factor for age-related diseases. Dietary restriction (DR) is the best-known non-invasive method to ameliorate aging in many organisms. However, the molecular mechanism and the signaling pathways that drive inflammaging across different tissues and how they are modulated by DR are not yet understood. Here we identify a multi-tissue gene network regulating inflammaging. This network is characterized by chromatin opening and upregulation in the transcription of innate immune system receptors and by activation of interferon signaling through interferon regulatory factors, inflammatory cytokines, and Stat1-mediated transcription. DR ameliorates aging-induced alterations of chromatin accessibility and RNA transcription of the inflammaging gene network while failing to rescue those alterations on the rest of the genome. Our results present a comprehensive understanding of the molecular network regulating inflammation in aging and DR and provide anti-inflammaging therapeutic targets.
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Envelhecimento , Inflamação , Envelhecimento/fisiologia , Cromatina , Humanos , Imunidade Inata , Inflamação/metabolismo , Interferons/metabolismo , Receptores Imunológicos/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA-the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP-TET interplay. RESULTS: Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. CONCLUSIONS: According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine.
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Poli ADP Ribosilação , Inibidores de Poli(ADP-Ribose) Polimerases , DNA/metabolismo , Metilação de DNA , Reparo do DNA , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
BACKGROUND: Spatial topography of the cystic fibrosis (CF) lung microbiota is poorly understood in childhood. How best to sample the respiratory tract in children for microbiota analysis, and the utility of microbiota profiling in clinical management of early infection remains unclear. By comparison with bronchoalveolar lavage (BAL), we assessed the ability of induced sputum (IS) sampling to characterise the lower airway microbiota. METHODS: Sample sets from IS and two or three matched BAL compartments were obtained for microbiota analysis as part of the CF-Sputum Induction Trial (UKCRN_14615, ISRCTNR_12473810). Microbiota profiles and pathogen detection were compared between matched samples. RESULTS: Twenty-eight patients, aged 1.1-17.7 years, provided 30 sample sets. Within-patient BAL comparisons revealed spatial heterogeneity in 8/30 (27%) sample sets indicating that the lower airway microbiota from BAL is frequently compartmentalised in children with CF. IS samples closely resembled one or more matched BAL compartments in 15/30 (50%) sets, and were related in composition in a further 9/30 (30%). IS detected 86.2% of the Top 5 genera found across matched BAL samples. The sensitivity of IS to detect specific CF-pathogens identified in matched BAL samples at relative abundance ≥5% varied between 43 and 100%, with negative predictive values between 73 and 100%. CONCLUSIONS: Spatial heterogeneity of the lower airway microbiota was observed in BAL samples and presents difficulties for consistent lung sampling. IS captured a microbiota signature representative of the lower airway in 80% of cases, and is a straightforward, non-invasive intervention that can be performed frequently to aid pathogen diagnosis and understand microbiota evolution in children with CF.
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Fibrose Cística , Microbiota , Criança , Humanos , Escarro , Fibrose Cística/diagnóstico , Líquido da Lavagem Broncoalveolar , PulmãoRESUMO
Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.
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Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multi-step demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated CC bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjoined with an sp1 or sp2 system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions.
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DNA Glicosilases/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Animais , Citosina/metabolismo , DNA/metabolismo , Reparo do DNA/genética , Humanos , Hidroxilação , Camundongos , Naegleria/genética , OxirreduçãoRESUMO
DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1-/- cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.
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5-Metilcitosina/química , Neoplasias Colorretais/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Células-Tronco Embrionárias/química , Animais , Técnicas de Cultura de Células , Neoplasias Colorretais/ultraestrutura , Meios de Cultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/ultraestrutura , Epigênese Genética , Células HCT116 , Humanos , Camundongos , Análise de Componente Principal , Análise Espectral RamanRESUMO
One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~â¯30â¯kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~â¯30, 32â¯kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway.
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Begomovirus/fisiologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Replicação Viral , Sequência de Aminoácidos , DNA Viral , Modelos Moleculares , Fosforilação , Doenças das Plantas/virologia , Conformação Proteica , Isoformas de ProteínasRESUMO
Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ácidos Nucleicos/isolamento & purificação , Animais , DNA/isolamento & purificação , Humanos , Campos Magnéticos , Microesferas , RNA/isolamento & purificaçãoRESUMO
The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology ( Oberacker et al., 2019 ). Updated protocols can be found on the associated webpage (https://bomb.bio).
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The encapsulation of folded proteins in stabilizing matrices is one of the challenges of soft-matter materials science. Capturing such fragile bio-macromolecules from aqueous solution, and embedding them in a lattice that stabilizes them against denaturation and decomposition is difficult. Here, we report that tetrahedral oligonucleotide hybrids as branching elements, and connecting DNA duplexes with sticky ends can assemble into materials. The material-forming property was used to capture DNA-binding proteins selectively from aqueous protein mixtures. The three-dimensional networks also encapsulate guest molecules in a size-selective manner, accommodating proteins up to a molecular weight of approximately 159â kDa for the connecting duplex lengths tested. Exploratory experiments with green fluorescent protein showed that, when embedded in the DNA-based matrix, the protein is more stable toward denaturation than in the free form, and retains its luminescent properties for at least 90â days in dry form. The noncrystalline biohybrid matrices presented herein may be used for capturing other proteins or for producing functional materials.
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SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.