Decoding transcriptional burst kinetics with BARe-seq

Multicellular organisms, from humans to flies, are made up of many individual cells. Although all the cells in one organism contain nearly identical genetic information, they form hundreds of different cell types, each performing unique functions; For example, a liver cell performs completely different tasks compared to a cell residing in the brain. The difference between the cells is therefore not their genetic information but which genes are active or inactive in each cell. The first determinant for a gene being in the active or inactive state is whether a gene is transcribed, which is the molecular process during which the cell reads out the genomic information and produces varying RNA and protein levels accordingly. The process transcribing genes from DNA into RNA is a sporadic, discontinuous cellular process that occurs in so called transcriptional bursts. These bursts vary in size (how much is made) and frequency (how often they occur) and are a key source of variation between cells. By adjusting the size or frequency of these bursts, cells can finely tune how genes are expressed. This process is controlled by special regulatory DNA sequences and by proteins called transcription factors (TFs) and cofactors (COFs). However, it is still unclear how these bursts are encoded in the DNA sequence and how specific sequence features and proteins work together to shape them. Previous research on the regulation of transcriptional bursts has been limited because only a small number of genes or sequences could be studied at a time. To address this challenge, I developed bulk allele resolution sequencing (BARe-seq), a new method that uses massively parallel reporter assays (MPRAs) to test up to 1,000 DNA sequences in a single experiment. This breakthrough allows for a detailed analysis of how transcriptional bursts are regulated. By focusing on transcriptional bursts the base unit of gene expression - this research will uncover new insights into how genes are regulated. It will help explain how cells with the same DNA can behave so differently and adapt to their roles and environments. This knowledge could have far-reaching implications for understanding health, disease, and cellular function.