Sub-chromosomal regulation of mitotic transcription inhibition

Chromosome condensation during mitosis has long been assumed to render mitotic chromatin incompatible with transcription. However, recent evidence shows that some chromosomal regions (e.g. centromeres, some genes) are transcribed in mitosis. These findings imply that mitotic transcription inhibition (MTI) is not a global event along the entire chromosome. Instead, I propose that mitotic transcriptional silencing is spatially regulated, allowing specific loci to evade MTI and maintain their transcription. Testing this novel hypothesis is challenging due to our poor understanding of the mechanisms driving MTI. Additionally, commonly used techniques to study transcription (e.g. genome-wide approaches) provide snapshots of the process but lack temporal resolution to dissect fast changes in transcription. With SUB-SCRIPT, I will implement an advanced imaging tool in live cells to uncover the dynamics of ongoing transcription throughout mitosis, for selected loci. I will apply this approach to selected genes to test how transcription regulation in mitosis varies depending on gene architecture/identity. In parallel, I will make use of a putative MTI factor (my preliminary observations) to manipulate the transcriptional state of selected genes during mitosis. Both strategies will capitalize on my vast expertise in dCas9 use for chromosome targeting, which I will further exploit to imaging RNAs (dCas13). Ongoing work in the host lab on chromosome assembly and novel mechanisms that drive MTI provides an excellent environment for project implementation. The completion of this project will uncover how different genes display unique kinetics of transcriptional regulation during mitosis and how MTI is regulated at the sub-chromosomal level, challenging current views on how mitotic transcription shut-down occurs. It will also provide novel tools and concepts to explore how MTI can actively impact mitotic fidelity, transcriptional control, cell physiology and potentially disease.