Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue

Abstract

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Chemical Expansion of the Methyltransferase Reaction: Tools for DNA Labeling and Epigenome Analysis

“…The availability of a new type of epigenomic information (Dnmt-selective methylation profiles) will facilitate the resolution of many puzzles of how genomic methylation is established and maintained during development, senescence, and disease. In another vein, we found that the deposited tags can render nucleosome repositioning in DNA, 69 which opens new avenues in manipulating epigenetic processes in live cells.…”

Biochemical and cellular insights into the Baz2B protein, a non-catalytic subunit of the chromatin remodeling complex

“…To analyse DNA binding of recombinant AT Baz2B -hooks and BRD BRG1 and BRD Baz2B , EMSAs were performed as described in ( 21 ) and ( 61 ), respectively. To test bromodomain binding to DNA, the Widom 147 bp DNA substrate was used and it was prepared as described ( 62 ).…”

5-Hydroxymethylcytosine: the many faces of the sixth base of mammalian DNA

“…[126][127][128][129][130] There is a general tendency for reduced twist and increased roll angles when C is substituted by mC or hmC, probably due to a steric hindrance of the cytosine 5-substituent. 129 hmC results in a 0.8 Å (4.5%) widening of the major groove at the site of modification, 127 similarly to mC or other C5 modifications, 131 reviewed in. 132 The hydroxymethyl moiety extends into the major groove (Fig.…”

“…129,136 Finally, there can be differences between effects of solitary hmC modifications and their cooperative impact in larger clusters. 129,131…”

“…129,136 Finally, there can be differences between effects of solitary hmC modifications and their cooperative impact in larger clusters. 129,131 Among non-canonical DNA structures, G-quadruplexes (G4) have emerged as a significant player in regulation of gene expression, replication and other genome metabolic processes. G4s are shown to form not only in telomeric regions but also throughout the genome in promoters, replication start sites, 138 therefore partly overlapping with methylation sites.…”