Abstract
Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and lens differentiation-specific α-, β-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.
Highlights
Studies of nascent transcription in a range of cell lines, primary cells, and a few tissues have shown that it occurs in bursts, i.e. oscillating between periods of activity and inactivity [1,2,3]
It has been shown that individual cells modulate transcriptional output by regulating the number of active phases of transcription (“burst fraction”) and/or the number of nascent RNA molecules produced within a burst (“burst size”); the duration of active and inactive phases of transcription (“burst duration”) is another quantitative parameter [10]
The burst fraction is related to the proportion of time each transcription sites (TSs) transcribes the gene when live cell recordings are conducted or to the proportion of active alleles per multiple individual nuclei when nascent transcription is analyzed by RNA fluorescent in situ hybridization (FISH) in fixed cells or tissues [5, 11, 12]
Summary
Studies of nascent transcription in a range of cell lines, primary cells, and a few tissues have shown that it occurs in bursts, i.e. oscillating between periods of activity and inactivity [1,2,3]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The burst fraction is related to the proportion of time each TS transcribes the gene when live cell recordings are conducted or to the proportion of active alleles per multiple individual nuclei when nascent transcription is analyzed by RNA FISH in fixed cells or tissues [5, 11, 12]. A number of studies investigated bursting parameters in unicellular organisms and cultured cells, very little is known about how cellular differentiation in mammalian tissues is regulated by transcriptional bursting [11,12,13]. How individual cells and tissues manage their transcriptional outputs during in vivo cellular differentiation to control burst fraction and size remains unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
