Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex

Abstract

SummaryDNA methylation at selective cytosine residues (5mC) and their removal by TET-mediated DNA demethylation are critical for setting up pluripotent states in early embryonic development 1-2 . TET enzymes successively convert 5mC to 5hmC, 5fC, and 5caC, the latter two of which are subject to removal by thymine DNA glycosylase (TDG) in conjunction with base excision repair [1][2][3][4][5][6] . Early reports indicate that 5fC and 5caC could be stably detected on enhancers, promoters, and gene bodies with distinct effects on gene expression, but the mechanisms have remained elusive 7,8 . Here we determined the X-ray crystal structure of elongating Pol II in complex with DNA template containing oxidized 5-methylcytosines (oxi-mCs), revealing specific hydrogen bonds between the 5-carboxyl group of 5caC and the conserved epi-DNA recognition loop in the polymerase. This causes a positional shift for incoming NTP thus compromising nucleotide addition. To test the in vivo significance of this structural insight, we determined the global effect of increased 5fC/5caC levels on transcription, finding that such DNA modifications indeed retarded Pol II elongation on gene bodies. These results demonstrate the functional impact of oxi-mCs on gene expression and suggest a novel role for Pol II to function as a specific and direct epigenetic sensor during transcription elongation.Epigenetic DNA methylation (5mC) is an important regulator of gene transcription recognized by several families of protein readers, such as methyl-CpG-binding domain proteins (MBDs) and ubiquitin-like PHD and RING finger domain containing proteins Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

RNA polymerase II acts as a selective sensor for DNA lesions and endogenous DNA modifications

“…Pol II is able to recognize 5caC via a specific hydrogen bond interaction between the carboxyl group of 5caC and a Pol II residue and slow down pol II transcription elongation. 26 It is expected that 5fC and 5fU may have similar interactions with pol II (Fig. 1).…”

“…Like 5caC, 5fC may form the similar hydrogen bond interaction with pol II through its carbonyl group as revealed by our modeling study. 26 Apart from the well-known function that Pol II acts as a key enzyme for synthesizing protein-coding and non-coding RNA transcripts from non-damaged templates, emerging evidence reveals that pol II may have additional functional roles. Pol II can serve as a specific sensor for DNA lesion attack and signal for a variety of DNA damage response pathways.…”

“…2C). 26 We renamed this loop as the epi-DNA recognition loop for it can recognize the modified cytosine in the major groove of the template strand. These specific hydrogen bonds cause misalignment of the 5caC template and subsequently disrupt proper alignment of the GTP substrate and 3 0 -RNA terminus, and result in a partially open conformation of the TL.…”

“…25 Further comparison of GRO-seq results of transcription elongation of WT (wild type) and TDG-KO (thymine DNA glycosylase -knock out) cell lines also support a negative correlation of the pol II elongation rate and endogenous 5fC/5caC levels, which is in good agreement with in vitro transcription results. 26 To further investigate the structural basis of how pol II recognizes oxi-mCs, we solved the structure of a pol II elongation complex containing a 5caC at the iC1 site (Fig. 2A).…”

RNA Pol II as a sensor of 5caC

“…In a recent study published in Nature, Wang et al [7] reconstituted yeast elongating RNA polymerase II (Pol II) complexed with a DNA template containing 5caC. In the structure determined at 3.3 Å resolution, 5caC adopts two configurations: a midway conformer and an insert conformer.…”