N⁴-acetyldeoxycytosine DNA modification marks euchromatin regions in Arabidopsis thaliana

Direct analogs of chemically modified bases that carry important epigenetic information, such as 5-methylcytosine (m5C)/5-methyldeoxycytosine (5mC), 5-hydroxymethylcytosine (hm5C)/5-hydroxymethyldeoxycytosine (5hmC), and N⁶-methyladenosine (m6A)/N⁶-methyldeoxyadenosine (6mA), are detected in both RNA and DNA, respectively. The modified base N⁴-acetylcytosine (ac4C) is well studied in RNAs, but its presence and epigenetic roles in cellular DNA have not been explored.

Here, we demonstrate the existence of N⁴-acetyldeoxycytosine (4acC) in genomic DNA of Arabidopsis with multiple detection methods. Genome-wide profiling of 4acC modification reveals that 4acC peaks are mostly distributed in euchromatin regions and present in nearly half of the expressed protein-coding genes in Arabidopsis. 4acC is mainly located around transcription start sites and positively correlates with gene expression levels. Imbalance of 5mC does not directly affect 4acC modification. We also characterize the associations of 4acC with 5mC and histone modifications that cooperatively regulate gene expression. Moreover, 4acC is also detected in genomic DNA of rice, maize, mouse, and human by mass spectrometry.

Our findings reveal 4acC as a hitherto unknown DNA modification in higher eukaryotes. We identify potential interactions of this mark with other epigenetic marks in gene expression regulation.