4sUDRB-sequencing for genome-wide transcription bursting quantification in breast cancer cells

Abstract

Epigenetics maintains cell-identity specific gene-expression patterns. However, within a population of isogenic cells of the same identity, a substantial variability in gene expression and responsiveness is still observed. Transcription bursting is a substantial source of this gene-expression variability or ‘noise’, contributing to phenotypic heterogeneity and potentially driving both physiological and pathological processes such as differentiation or tumorigenesis and drug resistance. Identification of transcription-bursting dynamics at a genome-wide scale has been restricted to inferring bursts in mRNA production computationally from the heterogeneity of mRNA levels in single cell transcriptomic data. Systematic characterisation of the genomic and epigenetic chromatin context of genes with defined transcription bursting behaviour has been incomplete. Here, we measured the bursting of transcription itself by genome-wide nascent RNA sequencing of breast cancer MCF-7 cells upon synchronisation of transcription with a transcription elongation inhibitor and by calibration using live cell imaging of nascent PP7-tagged GREB1 transcription. Comparing across the entire genome, we find transcription bursting to be ubiquitous, with burst sizes of up to 160 transcripts. Transcription bursting attributes ~85% to a trend in the variation in steady state gene expression between genes, whereas both burst frequency and nascent transcript degradation attribute minimally. Individual genes deviate strongly from this trend and engage both in anomalous burst size and frequency. We find that the presence of the TATA box or Inr sequence within gene promoters significantly predicts a larger burst size, as do promoter-associated YY1 and E2F1 transcription-factor binding motifs. Enrichment of the transcription start site with epigenetic marks such as H3K79me2 and H3Kl4ac is also strongly associated with the transcription burst size. Finally, we show that in these MCF-7 breast-cancer cells, genes with a larger transcription burst size exhibit a larger immediate transcriptional response following endocrine drug treatment. Our genome-wide transcription-bursting analysis method paves the way to elucidate the dynamic role of epigenetic regulation on dynamic transcription in pathophysiology.

The genome in space and time comes of age

“…They investigate the relationship between the transcription burst size and frequency of genes and their epigenetic regulation in MCF7 breast cancer cells. Simultaneously, they assess the functional impact of transcription bursting on cell behavior [ 24 ].…”

Heterochromatin Networks: Topology, Dynamics, and Function (a Working Hypothesis)

“…The stress response of Zaidela ascites carcinoma cells (washed and incubated in PBS in a plugged vial), seen as a few ~90 min collective waves of mRNA synthesis increased up to two to three times, was registered along with a bi-stable switch for the increased superhelicity of DNA opposed by the emergence and subsequent repair of DNA single-strand-breaks; these oscillations further faded and led to cell death [ 13 , 150 ]. MCF7 cells synchronized with the inhibitor interrupting mRNA elongation were recently examined by 4sUDRB-sequencing for genome-wide transcription [ 151 ]. The collective burst of 11,644 genes (each varying in intensity) was registered between 5 and 20 min, with a lower phase lasting until 40–50 min.…”