Critical DNA Binding Interactions of the Insulator Protein CTCF

Renda, Mario; Baglivo, Ilaria; Burgess-Beusse, Bonnie; Esposito, Sabrina; Fattorusso, Roberto; Felsenfeld, Gary; Pedone, Paolo V.

doi:10.1074/jbc.m706213200

Cited by 145 publications

(90 citation statements)

References 29 publications

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“…Methylated oligonucleotides targeting an intronic region of peroxisomal membrane protein 4 ( PXMP4 or PMP24 ) resulted in a single methylation mark on chromosomal DNA that downregulated gene expression relative to controls; this result corroborated differences observed between normal tissues and tumor cells [19]. Electromobility shift assays show that methylation at a single site impairs the binding of the genomic insulator CTCF [20].…”

Section: Introductionmentioning

confidence: 68%

Directed Evolution of Improved Zinc Finger Methyltransferases

Chaikind

Ostermeier

2014

PLoS ONE

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The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80%) and nearly unobservable levels of methylation at a off-target sites (<1%), as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences.

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Section: Introductionmentioning

confidence: 68%

Directed Evolution of Improved Zinc Finger Methyltransferases

Chaikind

Ostermeier

2014

PLoS ONE

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“…The presence of methyl groups enhanced binding affinity modestly in each case. In contrast to some proteins such as Myc (43,44), CTCF (45,46), STAT1 (47) and CREB (48) whose DNA-binding is inhibited by methylation, CpG-methylation does not reduce binding by normal WT1 or the three mutants, but instead either increases it somewhat or has no effect (Figure 3B–D). …”

Section: Resultsmentioning

confidence: 94%

Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

Hashimoto

Zhang

Zheng³

et al. 2016

Nucleic Acids Res

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Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences.

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“…These structures will then have lower energy and therefore higher probability of presence in the chromatin population. We calculated the distribution of the chromatin chains with such binder interactions, in which the binding energy of connecting two interacting sites is assigned to be 6 k B T (1240) (see Supplementary Information). This allows us to assess the scaling properties of different populations of chromatin chains under different looping conditions.…”

Section: Resultsmentioning

confidence: 99%

Spatial confinement is a major determinant of the folding landscape of human chromosomes

Gürsoy

Kenter

et al. 2014

Nucleic Acids Res

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The global architecture of the cell nucleus and the spatial organization of chromatin play important roles in gene expression and nuclear function. Single-cell imaging and chromosome conformation capture-based techniques provide a wealth of information on the spatial organization of chromosomes. However, a mechanistic model that can account for all observed scaling behaviors governing long-range chromatin interactions is missing. Here we describe a model called constrained self-avoiding chromatin (C-SAC) for studying spatial structures of chromosomes, as the available space is a key determinant of chromosome folding. We studied large ensembles of model chromatin chains with appropriate fiber diameter, persistence length and excluded volume under spatial confinement. We show that the equilibrium ensemble of randomly folded chromosomes in the confined nuclear volume gives rise to the experimentally observed higher-order architecture of human chromosomes, including average scaling properties of mean-square spatial distance, end-to-end distance, contact probability and their chromosome-to-chromosome variabilities. Our results indicate that the overall structure of a human chromosome is dictated by the spatial confinement of the nuclear space, which may undergo significant tissue- and developmental stage-specific size changes.

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Critical DNA Binding Interactions of the Insulator Protein CTCF

Cited by 145 publications

References 29 publications

Directed Evolution of Improved Zinc Finger Methyltransferases

Directed Evolution of Improved Zinc Finger Methyltransferases

Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

Spatial confinement is a major determinant of the folding landscape of human chromosomes

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