Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process

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 ( 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 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.

Promoter-enhancer looping and shadow enhancers of the mouse αA-crystallin locus

“…Due to the short window of time in which it has to produce all proteins required for the life of the organism, lens is an advantageous model tissue in which to study transcriptional control. αA-crystallin in particular, with its extraordinarily high relative expression levels must be highly regulated to generate such massive quantities before fiber cell organelles are degraded (Brennan et al, 2018; Limi et al, 2018). Previous work has identified an αA-crystallin promoter fragment (−364 to +45) that can support expression of a linked CAT gene, but only in fiber cells of the lens beginning after E12.5 (Overbeek et al, 1985), 2 days following endogenous αA-crystallin expression initiates (Robinson and Overbeek, 1996).…”

“…Their high level of expression, along with their distinct spatial distribution is essential for the transparency and refractive function of the lens. Together with globin genes in erythrocytes and calcium channel subunit Cacna2d in neurons, the crystallins rank amongst the most highly expressed genes in mammalian tissues (Sun et al, 2015a) and serve as an advantageous model to study fundamental principles of transcription during cellular differentiation (Limi et al, 2018). The most abundant αA-crystallin is a small heat shock chaperone protein and represents up to 17% of all newborn mouse lens water-soluble proteins.…”

Bidirectional Analysis of Cryba4-Crybb1 Nascent Transcription and Nuclear Accumulation of Crybb3 mRNAs in Lens Fibers

“…A procedure to generate probes for detection of nascent transcripts by RNA FISH and for RNA FISH and hybridization and imaging in the lens is described elsewhere 29. Briefly, a probe library consisting of 12 to 48 probes of 20-bp length was constructed against the exon regions of the genes of interest.…”

“…A procedure for imaging, image processing, and image analysis of RNA FISH data in the lens tissue is described elsewhere 29. Briefly, three-dimensional image data were acquired using the Zeiss Axio Observer CLEM microscope (Carl Zeiss).…”

“…In differentiating lens fibers, transcription is restricted by the denucleation process imposing additional limitations on the efficiency of the transcription prior to nuclear degradation 29. Recent studies of nascent transcription in multiple model cells and tissues revealed that the transcriptional process occurs in bursts29–32 and is executed by convoys of 20 to 30 molecules of RNA polymerase 33. Indeed, ChIP-seq data identified RNA polymerase II all across the active crystallin loci in lens chromatin, including their 3′-UTRs 34.…”