The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy

Abstract

While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.

Transcriptionally active chromatin loops contain both ‘active’ and ‘inactive’ histone modifications that exhibit exclusivity at the level of nucleosome clusters

“…The chromatin clusters labelled for H3K36me3 show heterogenous morphology. The clusters appear to be larger than global histone labelled Y loop clusters (33), with more “fuzzy” edges, suggesting the presence of looping DNA emanating from the edges of the clusters. Interestingly, there appear to be clear runs of Y loop chromatin which have continuous enrichment for H3K36me3, as the clusters appear approximately 100 nm apart from one another, the same spacing as the general histone labelled Y loop chromatin.…”

“…Notably, the H3K4me3 labelling intensity generally does not directly follow the histone labelling density and the peaks of H3K4me3 intensity overlay rather weak histone labelling, suggesting that the H3K4 mark is associated with relatively disperse chromatin (quantification is presented below). Although the H3K4me3 mark has been specifically linked to transcription initiation (39), the Y loop labelling is clearly not tightly restricted to the sites of initiation which are thought to be close to the Y chromosome mass associated with the nucleolus (33). Instead, the chains of H3K4me3 labelled clusters emanating from the nucleolus indicate that the H3K4me3 mark extends well into the transcription unit.…”

“…The histone labelled chromatin cluster widths are as described previously (33), with a median of 52 nm, and an interquartile range (IQR) between 44 and 61 nm. H3K27me3 labelled chromatin had a median width of 59 nm, and an IQR between 49 and 72 nm.…”

“…For the histone modifications examined, the ‘active’ marks of H3K4me3 and H3K36me3 and the ‘inactive’ mark H3K27me3, we find they are strikingly non-uniform in their distribution within transcription loops. The chromatin architecture of the Y loops is generally a chain of nucleosome clusters that extend out from the Y chromosome on the periphery of the nucleolus into the central nucleoplasm (33). The active histone marks are predominantly found on loop chromatin close to the nucleolus whereas H3K27me3 is present in domains more generally along the loops.…”

“…To overcome this obstacle, we have exploited the large nuclei of Drosophila melanogaster primary spermatocytes and their Y loops; single fibre, transcriptionally active chromatin loops that emerge from the Y chromosome as several megabase long structures. We have previously visualised and quantified the chromatin structure of the Y loops at super-resolution, showing that they are organised as chains of nucleosome clusters, with an average cluster width of approximately 50 nm (33). This aligns with current opinion based on multiple complementary techniques that consider interphase chromatin in vivo to be arranged as a heterogenous, discontinuous structure of irregular clusters or ‘clutches’ as opposed to a continuous 30 nm fibre (34).…”