In eukaryotic cells, transcription by RNA polymerase II occurs in the context of chromatin, requiring the transcription machinery to navigate through nucleosomes as it traverses gene bodies. Recent advances in structural biology have provided unprecedented insights into the mechanisms underlying transcription elongation. This review presents a structural perspective on transcription through chromatin, focusing on the latest findings from high-resolution structures of transcribing RNA polymerase II-nucleosome complexes. I discuss how RNA polymerase II, in concert with elongation factors such as SPT4/5, SPT6, ELOF1, and the PAF1 complex, engages with and transcribes through nucleosomes. The review examines the stepwise unwrapping of nucleosomal DNA as polymerase advances, the roles of elongation factors in facilitating this process, and the mechanisms of nucleosome retention and transfer during transcription. This structural perspective provides a foundation for understanding the intricate interplay between the transcription machinery and chromatin, offering insights into how cells balance the need for genetic accessibility with the maintenance of genome stability and epigenetic regulation.

Transcription of yeast RNA polymerase II through nucleosomes requires the assistance of the histone chaperone FACT (facilitates chromatin transcription). Yet, how FACT modulates the nucleosomal mechanical barrier to affect the polymerase's elongation dynamics is poorly understood. Using high-resolution single-molecule optical tweezers, we show that FACT greatly decreases the magnitude of the barrier by favoring the unwrapping of DNA from the distal H2A-H2B dimer, which, in turn, weakens the contacts near the dyad, significantly reducing the enzyme's crossing time. We show that barrier crossing depends on the asymmetric flexibility of the nucleosome arms, an asymmetry we find across the genome. Mechanical unwrapping of Cy3-H2A nucleosomes reveals that FACT reduces their unwrapping force and stabilizes a hexasome-like intermediate that retains both labeled dimers during successive unwrapping cycles. This intermediate is also observed after transcription. In conclusion, FACT facilitates nucleosomal transcription by weakening the barrier and actively assisting the maintenance of nucleosomal integrity after enzyme passage.

In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. Here, we report the 3.0-angstrom cryo–electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrap

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely