Investigating the Molecular Mechanisms Underlying Transcriptional Burst Regulation

Eukaryotic transcription has long been viewed as a steady binary process, where factors accumulate on DNA to continuously transcribe or repress genes. However, more recent single cell studies have revealed that eukaryotic transcription is much more sporadic, wherein genes undergo intermittent periods of high RNA production (i.e. bursts) interspersed between long periods of transcriptional inactivity. Modulation of entry into the transcriptional burst has been shown to be a key means of transcriptional control, but it is not known which factors are bound irrespective of transcription and which are associated only during a transcriptional burst. We overcame this issue by specifically targeting nascent RNA transcripts (i.e. gene-specific intron sequences), which are found only at active transcription sites. We used click-amplifying FISH (clampFISH), a single-molecule fluorescent signal amplification method recently developed in our lab, to target well-characterized genomic loci like human FKBP5 and mouse Hbb-b1 and separate our fixed cells by nascent RNA levels using flow cytometry. This generated sorted populations that either were actively transcribing the target locus at the time of fixation or not. In the sorted populations, we will examine whether candidate transcription factors known to bind each target locus are bound either in tight association with or irrespective of a transcriptional burst. Similarly, we will measure nucleosomal occupancy around each locus and chromatin conformation within and outside of an active transcriptional burst. With these data, we will have the first direct assessment of what biochemical differences exist between the bursting and transcriptionally inactive states. These differences will guide future mechanistic studies of transcriptional burst generation, helping us to address a longstanding question regarding the unknown biochemical underpinnings of transcriptional bursting.