Eukaryotic gene compaction takes place at multiple levels to package DNA to chromatin and chromosomes. Two of the most fundamental levels of DNA packaging are at the nucleosome and dinucleosome stacks. The nucleosome is the basic gene-packing unit and is composed of DNA wrapped around a histone core. Nucleosomes stack with one another for further compaction of DNA. The first stacking step leads to dinucleosome formation, which is driven by internucleosomal interactions between various parts of two nucleosomes. Histone proteins are rich targets for post-translational modifications, some of which affect the structure of the nucleosome and the interactions between nucleosomes. These effects are often implicated in the regulation of various genomic transactions. In particular, histone H2B ubiquitylation has been associated with facilitated transcription and hexasome formation. Here, we employed semi-synthetically ubiquitylated histone H2B and single-molecule FRET to investigate the effects of H2B ubiquitylations at lysine 34 (H2BK34) and lysine 120 (H2BK120) on the structure of the nucleosome and the interactions between two nucleosomes. Our results suggest that H2BK34 ubiquitylation widens the DNA gyre gap in the nucleosome and stabilizes long-and short-range internucleosomal interactions while H2BK120 ubiquitylation does not affect the nucleosome structure or internucleosomal interactions. These results suggest potential roles for H2B ubiquitylations in facilitated transcription and hexasome formation while maintaining the structural integrity of chromatin.
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
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