RESUMO
5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel 'CpG duplex marks' could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.
Assuntos
5-Metilcitosina , DNA , Epigênese Genética , Animais , Ilhas de CpG/genética , DNA/química , Metilação de DNA , Epigenômica , Mamíferos/metabolismo , Conformação MolecularRESUMO
Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5-methylation (mC), a total of four different 5-modifications can theoretically co-exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5-carboxylcytosine (caC) dyads based on the methyl-CpG-binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC-dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design.
Assuntos
Citosina , Citosina/análogos & derivados , Metilação de DNA , Animais , Ilhas de CpG , Citosina/química , Epigênese Genética , Mamíferos/metabolismoRESUMO
RNA-binding proteins are essential regulators of RNA processing and function. Translational repression assays can be used to study how they interact with specific RNA sequences by insertion of such a consensus sequence into the 5' untranslated region of a reporter mRNA and measuring reporter protein translation. The straightforward set-up of these translational repression assays avoids the need for the isolation of the protein or the RNA providing speed, robustness and a low-cost method. Here, we report the optimization of the assay to function with linear RNA sequences instead of the previously reported hairpin type sequences to allow the study of a wider variety of RNA-binding proteins. Multiplication of a consensus sequence strongly improves the signal allowing analysis by both fluorescence intensity measurements and flow cytometry.
Assuntos
Biossíntese de Proteínas , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , DNA/isolamento & purificação , Proteína 2 de Ligação a Metil-CpG/química , Fragmentos de Peptídeos/química , DNA/química , Metilação de DNA , Evolução Molecular Direcionada , Células HEK293 , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Fragmentos de Peptídeos/genética , Domínios ProteicosRESUMO
Transcription-activator-like effectors (TALEs) are repeat-based, programmable DNA-binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging-based analysis of cellular 5-methylcytosine (5â mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co-stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C-selective repeat that is blocked by 5â mC, and a control TALE targeting the position with a universal repeat that binds both C and 5â mC. To enhance this approach, we report herein the development of novel 5â mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5â mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5â mC binders. Compared to the 5â mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5â mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co-stains of cellular SATIII DNA with a corresponding C-selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5â mC functions in chromatin regulation by inâ situ imaging with increased dynamic range.
Assuntos
5-Metilcitosina/análise , Metilação de DNA , Processamento de Imagem Assistida por Computador/métodos , Sondas Moleculares/metabolismo , Sequências Repetitivas de Ácido Nucleico , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Engenharia Genética , Células HEK293 , Humanos , Sondas Moleculares/química , Efetores Semelhantes a Ativadores de Transcrição/químicaRESUMO
Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
Assuntos
Genoma Fúngico , Biblioteca Genômica , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta , Proteoma/genética , Proteômica , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Sitios de Sequências RotuladasRESUMO
5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos da radiação , Luz , 5-Metilcitosina/metabolismo , Biocatálise , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Células HEK293 , Humanos , Mutação , Transcriptoma/efeitos da radiaçãoRESUMO
Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.
Assuntos
5-Metilcitosina/metabolismo , Dioxigenases/metabolismo , Humanos , Oxirredução , TranscriptomaRESUMO
We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation inâ situ with nucleotide, locus, and cell resolution.
Assuntos
5-Metilcitosina/metabolismo , Genômica , Imagem Molecular , Nucleotídeos/metabolismo , Células HeLa , Humanos , Análise de Célula ÚnicaRESUMO
5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.
Assuntos
Citosina/análogos & derivados , DNA/química , Efetores Semelhantes a Ativadores de Transcrição/química , Animais , Reagentes de Ligações Cruzadas/química , Citosina/análise , Citosina/química , Epigênese Genética , Genoma , Genômica/métodos , Humanos , Masculino , Camundongos , Fenilalanina/análogos & derivados , Fenilalanina/química , Reação em Cadeia da PolimeraseRESUMO
[This corrects the article DOI: 10.1039/D2MD00186A.].
RESUMO
Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins that can be used for sequence-specific, imaging-based analysis of cellular 5-methylcytosine. However, this has so far been limited to highly repetitive satellite DNA. To expand this approach to the analysis of coding single gene loci, we here explore a number of signal amplification strategies for increasing imaging sensitivity with TALEs. We develop a straightforward amplification protocol and employ it to target the MUC4 gene, which features only a small cluster of repeat sequences. This offers high sensitivity imaging of MUC4, and in costaining experiments with pairs of one TALE selective for unmethylated cytosine and one universal control TALE enables analyzing methylation changes in the target independently of changes in target accessibility. These advancements offer prospects for 5-methylcytosine analysis at coding, nonrepetitive gene loci by the use of designed TALE probe collections.
Assuntos
5-Metilcitosina , Efetores Semelhantes a Ativadores de Transcrição , Efetores Semelhantes a Ativadores de Transcrição/genética , 5-Metilcitosina/metabolismo , DNA/genética , DNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Proteínas de Ligação a DNA/metabolismoRESUMO
Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.
RESUMO
Modifications of the cytosine 5-position are dynamic epigenetic marks of mammalian DNA with important regulatory roles in development and disease. Unraveling biological functions of such modified nucleobases is tightly connected with the potential of available methods for their analysis. Whereas genome-wide nucleobase quantification and mapping are first-line analyses, targeted analyses move into focus the more genomic sites with high biological significance are identified. We here review recent developments in an emerging field that addresses such targeted analyses via probes that combine a programmable, sequence-specific DNA-binding domain with the ability to directly recognize or cross-link an epigenetically modified nucleobase of interest. We highlight how such probes offer simple, high-resolution nucleobase analyses in vitro and enable in situ correlations between a nucleobase and other chromatin regulatory elements at user-defined loci on the single-cell level by imaging.
Assuntos
5-Metilcitosina/química , DNA/química , Epigênese Genética/genética , Sítios de Ligação , Cromatina/química , Reagentes de Ligações Cruzadas/química , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Corantes Fluorescentes/química , Genômica , Humanos , Conformação Molecular , Imagem Molecular , Imagem Óptica , Análise de Célula ÚnicaRESUMO
Transcription-activator like effectors (TALEs) are DNA-binding proteins used for genome targeting. TALEs contain a central domain of concatenated repeats, of which each selectively recognizes one nucleobase at the DNA major groove. Based on this simple and predictable interaction with little context dependence, TALEs offer programmable targeting of user-defined DNA sequences. Since many epigenetic DNA modifications protrude into the DNA major groove, natural and engineered TALE repeats can provide "epigenetic" selectivity, making TALEs a flexible platform to design probes for the analysis of epigenetic DNA modifications. Here, we describe guidelines for the design of TALE proteins with selectivity for epigenetic cytosine 5-modifications, the validation of their interaction with modified DNA nucleobases, and their employment in affinity enrichment assays. These techniques enable quantification of epigenetic nucleobases in user-defined genomic DNA sequences with nucleotide and strand resolution.
Assuntos
Epigenômica/métodos , Efetores Semelhantes a Ativadores de Transcrição/síntese química , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , 5-Metilcitosina/química , Animais , Citosina/metabolismo , DNA/química , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/genética , Genoma/genética , Humanos , Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
5-Methylcytosine (mC) exists in CpG dinucleotides of mammalian DNA and plays key roles in chromatin regulation during development and disease. As a main regulatory pathway, fully methylated CpG are recognized by methyl-CpG-binding domain (MBD) proteins that act in concert with chromatin remodelers, histone deacetylases and methyltransferases to trigger transcriptional downregulation. In turn, MBD mutations can alter CpG binding, and in case of the MBD protein MeCP2 can cause the neurological disorder Rett syndrome (RTT). An additional layer of complexity in CpG recognition is added by ten-eleven-translocation (TET) dioxygenases that oxidize mC to 5-hydroxymethyl-, 5-formyl- and 5-carboxylcytosine, giving rise to fifteen possible combinations of cytosine modifications in the two CpG strands. We report a comprehensive, comparative interaction analysis of the human MBD proteins MeCP2, MBD1, MBD2, MBD3, and MBD4 with all CpG combinations and observe individual preferences of each MBD for distinct combinations. In addition, we profile four MeCP2 RTT mutants and reveal that although interactions to methylated CpGs are similarly affected by the mutations, interactions to oxidized mC combinations are differentially affected. These findings argue for a complex interplay between local TET activity/processivity and CpG recognition by MBDs, with potential consequences for the transcriptional landscape in normal and RTT states.
Assuntos
Ilhas de CpG , Citosina/análogos & derivados , Citosina/química , Repetições de Dinucleotídeos , Proteína 2 de Ligação a Metil-CpG/química , Síndrome de Rett , Citosina/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismoRESUMO
Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Marcação de Genes/métodos , Reação em Cadeia da Polimerase/métodos , Alelos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , Células Cultivadas , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Recombinação Homóloga , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Oligonucleotídeos/genética , RNA Guia de Cinetoplastídeos/genética , TransfecçãoRESUMO
Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Células Clonais , Biblioteca Gênica , Engenharia Genética , Genoma Fúngico , Proteínas de Fluorescência Verde/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Transcription-activator-like effectors (TALEs) are repeat-based proteins featuring programmable DNA binding. The repulsion of TALE repeats by 5-methylcytosine (5mC) and its oxidized forms makes TALEs potential probes for their programmable analysis. However, this potential has been limited by the inability to engineer repeats capable of actual, fully selective binding of an (oxidized) 5mC: the extremely conserved and simple nucleobase recognition mode of TALE repeats and their extensive involvement in inter-repeat interactions that stabilize the TALE fold represent major engineering hurdles. We evaluated libraries of alternative, strongly truncated repeat scaffolds and discovered a repeat that selectively recognizes 5-carboxylcytosine (5caC), enabling construction of the first programmable receptors for an oxidized 5mC. In computational studies, this unusual scaffold executes a dual function via a critical arginine that provides inter-repeat stabilization and selectively interacts with the 5caC carboxyl group via a salt-bridge. These findings argue for an unexpected adaptability of TALE repeats and provide a new impulse for the design of programmable probes for nucleobases beyond A, G, T and C.
RESUMO
The GRPr, highly expressed in prostate PCa and breast cancer BCa, is a promising target for the development of new PET radiotracers. The chelator HBED-CC ( N, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine- N, N'-diacetic acid) was coupled to the bombesin peptides: HBED-C-BN(2-14) 1, HBED-CC-PEG2-[d-Tyr6,ß-Ala11,Thi13,Nle14]-BN(6-14) 2, HBED-CC-Y-[d-Phe6,Sta13,Leu14]-BN(6-14) (Y = 4-amino-1-carboxymethylpiperidine) 3, and HBED-CC-{PEG2-Y-[d-Phe6,Sta13,Leu14]-BN(6-14)}2 4 (homodimer). Compounds 1-4 presented high binding affinities for GRPr (T47D, 0.56-3.51 nM; PC-3, 2.12-4.68 nM). In PC-3 and T47D cells, agonists [68Ga]1 and [68Ga]2 were mainly internalized while antagonists [68Ga]3 and [68Ga]4 were surface bound. Cell-related radioactivity reached a maximum after 45 min, while tracer levels followed GRPr expression (PC-3 > T47D > LNCaP > MDA-MB-231). [68Ga]4 showed the highest cell-bound radioactivity (PC-3 and T47D). In vivo, tumor (PC-3) targeting for [68Ga]3 and [68Ga]4 increased over time, with dynamic µPET showing clearer tumors images at later time points. [68Ga]3 and [68Ga]4 can be considered suitable PET tracers for imaging PCa and BCa expressing GRPr.