Transcriptional Output Transiently Spikes Upon Mitotic Exit

The pulsatile nature of gene activity has recently emerged as a general property of the transcriptional process. It has been shown that the frequency and amplitude of transcriptional bursts can be subjected to extrinsic regulation. Here we have investigated if these parameters were constant throughout the cell cycle using the single molecule RNA FISH technique. We found evidence of transcriptional spikes upon mitotic exit in three different human cell lines. Recording of cell growth prior to hybridization and immuno-RNA FISH analysis revealed that these spikes were short-lived and subsided before completion of cytokinesis. The transient post-mitotic increase in transcriptional output was found to be the result of cells displaying a higher number of active alleles and/or an increased number of nascent transcripts per active allele, indicating that both the burst fraction and the amplitude of individual bursts can be increased upon mitotic exit. Our results further suggest that distinct regulatory mechanisms are at work shortly after mitotic exit and during the rest of interphase. We speculate that transcriptional spikes are associated with chromatin decondensation, a hallmark of post-mitotic cells that might alter the dynamics of transcriptional regulators and effectors.


Preparation of metaphase spreads
Growing cells (HepG2 and HT-1080) were incubated in medium containing 0.1 g/ml colchicine for 20 minutes. Cells were then trypsinized, incubated in an hypotonic solution (0.56M KCl) for 12 minutes at 37ºC, and fixed with methanol:acetic acid (3:1) for 20 minutes at -20ºC. After several washes, fixed cells were dropped on microscope slides in a humidified chamber in a water bath at 55ºC and left to dry for 2 minutes. Metaphase spreads were aged at room temperature for at least 5 days.

FISH on metaphase spreads with BAC probes
Bacterial artificial chromosomes (BAC) comprising the human POLR2A gene (RP11-104H15, CHORI BACPAC Resource Center) or sequences 4 Mb centromeric to the human TFRC gene (3q28, RP11-183L23, a kind gift of Dr. Marion Cremer, Ludwig-Maximilians Universität München) were extracted from 1 ml of bacterial cultures and linearly amplified using the GenomiPhi V2 kit according to the manufacturer's instructions (GE Healthcare Life Sciences). Labeling of BAC DNA and FISH on metaphase spreads were performed as previously described 1 . Briefly, DNA was labeled either with DIG (POLR2A) or biotin (3q28). Before hybridization, samples were treated with pepsin at 100 g/ml in 0.01N for 8-9 minutes at room temperature, dehydrated and air-dried. Probes (at final concentration of 5 ng/l) and target DNA were denatured simultaneously by placing the slides on a hot block at 76ºC for 2 minutes. Hybridization was carried out at 37ºC for 2 days. After washes, hybridized probes were detected with antibodies against DIG coupled to Cy5 (POLR2A) or streptavidin coupled to Cy3 (3q28/TFRC). Samples were counterstained with DAPI and imaged on a Nikon TiE widefield fluorescence microscope.

Analysis of the mean DAPI intensity at transcriptional spots
We have quantified the DAPI signal intensity in the regions of transcriptional spots in the telophase/early G1 nuclei in the following way. 1) the z-section containing the focus of a given transcription spot was determined manually; 2) DAPI spot , the pixel value at the spot maxima was determined in the corresponding DAPI channel; 3) the DAPI pixel intensity values were determined in a region-of-interest of ~3 m x 3 m (50 pixels by 50 pixels) drawn around the transcriptional spot and distributed into 256 bins of equal size; 4) the bin comprising the DAPI spot value was noted. The above steps were repeated for 15 spots for each gene. Results are plotted separately for individual spots.

Immuno-RNA FISH to detect tubulin along with TFRC and POLR2A RNAs
HepG2 cells were grown on uncoated 18 mm x 18 mm #1.5 coverslips, fixed with 4% formaldehyde in 1X PBS for 10 minutes, washed and stored overnight in 70% EtOH at 4°C.
After rehydration in PBS, cells were blocked for 7 minutes in PBS containing 1 mg/ml bovine serum albumin and 10mM ribonucleoside vanadyl complex (New England Biolabs), which was also included in all antibody solutions during the subsequent immunostaining procedure.
Cells were then incubated in a 1:20 dilution (in PBS) of a mouse monoclonal antibody against -tubulin (clone E7, Developmental Studies Hybridoma Bank at the University of Iowa) for 30 minutes at room temperature. After brief washes with PBS (2 times, 2 minutes each), cells were incubated in a 1:200 dilution (in PBS, final concentration of 6.5 g/ml) of a secondary biotinylated goat anti-mouse antibody (Jackson Labs) for 30 minutes at room temperature.
After brief washes with PBS (3 times, 2 minutes each), the immunocomplexes were fixed for 10 minutes with 2% (v/v) formaldehyde in PBS. After successive washes with PBS (1 minute) and 2X SSC (2 minutes), followed by equilibration in 2XSSC/10% formamide (5 minutes), smRNA FISH was performed as described in Materials and Methods. After the last wash with 2X SSC, cells were further stained for 20 minutes at room temperature.with DAPI (1 g/ml) and a 1:400 dilution of avidin-Alexa 488 (final concentration of 2.5 g/ml, ThermoFisher/Life Technologies). Finally, samples were washed twice with 2X SSC and mounted in Prolong Gold.          (20-mer, from 5' to 3'), the oligonucleotide identification and number, its position relative to the first nucleotide of the intron and its GC contents. The Ensembl Gene ID for human POLR2A is ENSG00000181222.