Cryo-EM structures of RNA polymerase II-nucleosome complexes rewrapping transcribed DNA.

RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.

Structural Basis of Damaged Nucleotide Recognition by Transcribing RNA Polymerase II in the Nucleosome.

In transcription-coupled repair (TCR), transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits TCR proteins to the damaged site. However, the mechanism by which RNAPII recognizes a DNA lesion in the nucleosome remains enigmatic. In the present study, we inserted an apurinic/apyrimidinic DNA lesion analogue, tetrahydrofuran (THF), in the nucleosomal DNA, where RNAPII stalls at the SHL(-4), SHL(-3.5), and SHL(-3) positions, and determined the structures of these complexes by cryo-electron microscopy. In the RNAPII-nucleosome complex stalled at SHL(-3.5), the nucleosome orientation relative to RNAPII is quite different from those in the SHL(-4) and SHL(-3) complexes, which have nucleosome orientations similar to naturally paused RNAPII-nucleosome complexes. Furthermore, we found that an essential TCR protein, Rad26 (CSB), enhances the RNAPII processivity, and consequently augments the DNA damage recognition efficiency of RNAPII in the nucleosome. The cryo-EM structure of the Rad26-RNAPII-nucleosome complex revealed that Rad26 binds to the stalled RNAPII through a novel interface, which is completely different from those previously reported. These structures may provide important information to understand the mechanism by which RNAPII recognizes the nucleosomal DNA lesion and recruits TCR proteins to the stalled RNAPII on the nucleosome.

Essential roles of Windei and nuclear monoubiquitination of Eggless/SETDB1 in transposon silencing.

Eggless/SETDB1 (Egg), the only essential histone methyltransferase (HMT) in Drosophila, plays a role in gene repression, including piRNA-mediated transposon silencing in the ovaries. Previous studies suggested that Egg is post-translationally modified and showed that Windei (Wde) regulates Egg nuclear localization through protein-protein interaction. Monoubiquitination of mammalian SETDB1 is necessary for the HMT activity. Here, using cultured ovarian somatic cells, we show that Egg is monoubiquitinated and phosphorylated but that only monoubiquitination is required for piRNA-mediated transposon repression. Egg monoubiquitination occurs in the nucleus. Egg has its own nuclear localization signal, and the nuclear import of Egg is Wde-independent. Wde recruits Egg to the chromatin at target gene silencing loci, but their interaction is monoubiquitin-independent. The abundance of nuclear Egg is governed by that of nuclear Wde. These results illuminate essential roles of nuclear monoubiquitination of Egg and the role of Wde in piRNA-mediated transposon repression.