Active RNA polymerases are localized within discrete transcription ‘factories’ in human nuclei

Abstract

Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30–2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription ‘factories’. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.

Live imaging of transcription sites using an elongating RNA polymerase II–specific probe

“…The localization of RNAP2 and its phosphorylated forms in the cell nucleus has been analyzed by light and electron microscopy. RNAP2 Ser2ph is closely associated with nascent RNA labeled with bromo-UTP, which is consistent with this modification being a mark of elongating form ( Iborra et al, 1996 ; Pombo et al, 1999 ). From the number of RNAP2 transcription sites (∼8,000) and actively transcribing molecules (∼65,000) in a HeLa cell, it was estimated that one transcription site with ∼80-nm diameter contains eight RNAP2 molecules, on average, to form transcription “factories” ( Jackson et al, 1998 ; Cook, 1999 ).…”

“…Although it is still possible to have tens of mintbodies per RNAP2 exhibit a fluorescence focus, the number of foci in a HeLa cell does not correspond to the number of total active RNAP2 molecules, which is estimated to be tens of thousands ( Jackson et al, 1998 ; Kimura et al, 1999 ). The maximum number of foci detected (∼5,000) was rather similar to several thousands of transcription sites containing multiple transcription units, suggesting that the mintbody foci likely correspond to transcription factories ( Iborra et al, 1996 ; Pombo et al, 1999 ; Cook, 1999 ).…”

“…As one CTD harbors 52 repeats and more than half of Ser2 in the repeats can be phosphorylated ( Schüller et al, 2016 ), a focus may be derived from a single RNAP2 molecule that is bound with multiple RNAP2 Ser2ph-mintbodies. However, it is also possible that a focus may contain multiple polymerases ( Iborra et al, 1996 ). The size of the foci was estimated to be ∼200 nm ( Fig.…”

Long-range chromosomal interactions and gene regulation

“…Each cell only contains a limited number of transcription factories, and at any factory multiple genes are engaged in transcription at any given point in time. 23,27 Direct, but apparently non-specific associations between active genes could be detected using a 3C-based approach. 25 These associations likely occur as a result of the association of genes with the same transcription factory, which brings these genes in close spatial proximity.…”

Robust and efficient gene regulation through localized nuclear microenvironments

“…Perhaps one of the first observations that transcription does not occur homogeneously inside the nucleus was the visualization of discrete foci of nascent RNAs and RNA polymerases, and their overlap, leading to the concept of 'transcription factories' (Glossary, Box 1) (Bregman et al, 1995;Iborra et al, 1996;Jackson et al, 1993Jackson et al, , 1998Wansink et al, 1993). Further experiments suggested that the movement of genes into or out of transcription factories results in an increase or reduction of transcription (Osborne et al, 2004;Rieder et al, 2012).…”