BackgroundDNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.ResultsWe compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex).Conclusions0ur screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.

SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5′-GGGCGG-3′, we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells.

The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered as the “seventh” base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase (TDG). Genomics and proteomics studies have suggested a further role for 5fC in transcription regulation through chromatin remodeling. Herein we propose how 5fC might signal these processes through its effect on DNA conformation. Biophysical and structural analysis revealed that 5fC alters the structure of the DNA double helix leading to a conformation unique amongst known DNA structures including those comprising other cytosine modifications. The 1.4 Å resolution X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shown how 5fC changes the geometry of the grooves and base pairs associated with the modified base, which lead to helical under-winding.

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It all clicks into place: A potent telomere‐targeting small molecule has been identified by using the copper‐free 1,3‐dipolar cycloaddition of a series of alkyne and azide building blocks catalyzed by a non‐Watson–Crick DNA secondary structure (see picture). This method rapidly identifies, otherwise unanticipated, potent small‐molecule probes to selectively target a given RNA or DNA.

Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information. The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine (5fC) is linked to tissue-specific nucleosome organization. Our study reveals that 5fC is associated with increased nucleosome occupancy in vitro and in vivo. We demonstrate that 5fC-associated nucleosomes at enhancers in mammalian hindbrain and heart are linked to elevated gene expression. Our study also reveals the formation of a reversible-covalent Schiff base linkage between lysines of histone proteins and 5fC within nucleosomes in a cellular environment. We define their specific genomic loci in mouse embryonic stem cells and look into the biological consequences of these DNA-histone Schiff base sites. Collectively, our findings show that 5fC is a determinant of nucleosome organization and plays a role in establishing distinct regulatory regions that control transcription. A series of in vitro and in vivo studies has now shown that 5fC is linked to increased nucleosome occupancy and stability. Moreover, there is evidence that Schiff base formation between histones and 5fC impacts RNA polymerase II transcription activity in mouse embryonic stem cells.

Chemically modified bases naturally exist in genomic DNA. Research into these bases has been invigorated by the discovery of several modified bases in the mammalian genome, in particular the oxidised derivatives of 5methylcytosine, such as 5-hydroxymethylcytosine and 5-formylcytosine, as well as the enzymes that form and process them, such as DNA methyltransferases (DNMTs) and the ten-eleven translocation (TET) enzymes. In this Review we provide an overview of natural, modified bases that have been reported in DNA, our current knowledge of their roles, and the techniques that have enabled us to probe their functions. Analytical methods have been invaluable in helping advance this field. For example, chemical and enzymatic methods have provided the means to detect and decode modified bases, giving rise to an expanding array of sequencing approaches.Advanced liquid chromatography and tandem mass spectrometry have provided the means to detect and quantify modified bases with very high sensitivity, increasing the prospects for the discovery of unknown modifications. It is already evident that natural, modified DNA bases and their associated enzymology are of fundamental importance to normal biology and to disease. The next decade promises to yield more insights, discoveries and impact from this burgeoning field of research.

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.