Nucleosomes are the basic unit of chromatin that ensure genome integrity and control access to the genetic information. The organization of not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/224444 doi: bioRxiv preprint first posted online Nov. 24, 2017; The organization of nucleosomes is an important feature to the chromatin structure. The DNA sequence itself has been shown to be a determinant of nucleosome positioning raising questions about the effect of DNA modifications on chromatin structure. 5-Formylcytosine is generated by the oxidation of 5mC by TET enzymes. It can undergo base excision repair by the thymine DNA glycosylase (TDG) (5) and it is therefore thought to mark sites that undergo active demethylation (6). However, we have previously shown that the 5fC distribution not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/224444 doi: bioRxiv preprint first posted online Nov. 24, 2017; 3 was tissue-dependent in mice (7) and that 5fC was a stable modification in genomic DNA in vivo (8). Additionally we and others have demonstrated that 5fC can alter the physical properties of the DNA double helix (9, 10) and the identification of 5fC-binding chromatin remodelers and transcription factors in vitro (11,12) raises fundamental questions about its involvement in chromatin biology. Moreover, the nature of the formyl group confers specific chemical properties to 5fC that are unique from other cytosine modifications such as 5-hydroxymethylcytosine (5hmC) and 5-carboxycytosine (5caC). We therefore hypothesized that 5fC may be important in nucleosomal organization, particularly during early development, where increased levels of 5fC have been reported (8).To test this hypothesis, we assessed the impact of 5fC on nucleosome core particles formation and stability. We carried out biochemical studies on three different DNA sequences, Widom 601, MMTV-A and 5SrDNA with varying CpG and GC content ( Fig. 1A and Supplementary Table 1). All sequences are able to form nucleosomes of different stabilities and properties and have been extensively characterized (13-16). In addition, effects into 5-methylcytosine (5mC) (17, 18) and 5-hydroxymethylcytosine (5hmC) (19) on nucleosome occupancy have been investigated on the Widom 601 sequence, however it is unclear how 5fC affects nucleosome formation and stability. To address this, we compared nucleosome occupancy after chaperone-mediated assembly using DNA with unmodified and modified cytosines. We observed that 5mC, 5hmC and 5fC each caused an increase in nucleosome occupancy compared to unmodified Widom 601 DNA (Fig. 1A), with 5fC-DNA significantly (unpaired t-test, p value ≤ 0.0001) displaying the strongest effect (Fig. 1B). Notably we found that only 5fC not peer-reviewed) is the author/funder. All rights reserved. No reuse a...