RESUMEN
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.
Asunto(s)
5-Metilcitosina , ADN , Epigénesis Genética , Animales , Islas de CpG/genética , ADN/química , Metilación de ADN , Epigenómica , Mamíferos/metabolismo , Conformación MolecularRESUMEN
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.
Asunto(s)
Citosina , Citosina/análogos & derivados , Metilación de ADN , Animales , Islas de CpG , Citosina/química , Epigénesis Genética , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , ADN/aislamiento & purificación , Proteína 2 de Unión a Metil-CpG/química , Fragmentos de Péptidos/química , ADN/química , Metilación de ADN , Evolución Molecular Dirigida , Células HEK293 , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Fragmentos de Péptidos/genética , Dominios ProteicosRESUMEN
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.
Asunto(s)
Genoma Fúngico , Biblioteca Genómica , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura Abierta , Proteoma/genética , Proteómica , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Lugares Marcados de SecuenciaRESUMEN
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.
Asunto(s)
5-Metilcitosina , Efectores Tipo Activadores de la Transcripción , Efectores Tipo Activadores de la Transcripción/genética , 5-Metilcitosina/metabolismo , ADN/genética , ADN/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas de Unión al ADN/metabolismoRESUMEN
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.
Asunto(s)
Islas de CpG , Citosina/análogos & derivados , Citosina/química , Repeticiones de Dinucleótido , Proteína 2 de Unión a Metil-CpG/química , Síndrome de Rett , Citosina/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismoRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Marcación de Gen/métodos , Reacción en Cadena de la Polimerasa/métodos , Alelos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Línea Celular , Células Cultivadas , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Genes Reporteros , Secuenciación de Nucleótidos de Alto Rendimiento , Recombinación Homóloga , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Oligonucleótidos/genética , ARN Guía de Kinetoplastida/genética , TransfecciónRESUMEN
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.