Transcription condensates compartmentalize essential components of the transcription machinery, including RNA polymerase II, transcription factors, coactivators, mediator, and Brd4. Condensates associate with highly active chromatin regions characterized by stereotypical epigenetic histone modifications. H3K27ac, along with H3K4me1 and H3K4me3, characterizes active enhancer and promoter regions, respectively, whereas transcriptionally inactive heterochromatin is characterized by H3K27me3. Using conventional fluorescence microscopy, we show that all three active chromatin marks are enriched at transcription condensates. The inactive H3K27ac mark is depleted from condensates. To investigate the organization of functional chromatin around condensates with higher precision, we utilize multicolor 3D single-molecule localization microscopy. Our data reveal that H3K27ac and H3K4me3 chromatin marks partition into transcription condensates. In contrast, the H3K4me1 mark is most abundant at the condensate surface. Segmentation of epigenetic nanoclusters suggests that multiple active regulatory elements interact with individual condensates simultaneously. Accordingly, we find several distinct nanoclusters of Ser2p-phosphorylated Pol II associated with individual condensates. Together, our results suggest that condensates harbor a promoter-rich central region that is decorated with enhancer marks. To uncover the mechanisms that lead to differential chromatin organization at condensates, we employed drug treatments. Transcription initiation inhibition with triptolide (TPL), accompanied by Pol II degradation, resulted in elevated levels of all three active chromatin marks at condensates, whereas inhibiting Brd4 binding to H3K27ac with JQ1 resulted in partial loss of H3K27ac signal at condensates. H3K4me1/3 signal was largely unaffected by JQ1 treatment. These results indicate that chromatin interactions with the condensate constituents Pol II and Brd4 play a minor role in chromatin compaction at condensates. We speculate that active transcription counteracts chromatin compaction at condensates by chromatin remodeling proteins.
Pandey et al. (Sun,) studied this question.